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Funder: Wessex Medical Research, Funder: Health Education England, Funder: Rosetrees Trust, BACKGROUND: Genomic variants which disrupt splicing are a major cause of rare genetic diseases. However, variants which lie outside of the canonical splice sites are difficult to interpret clinically. Improving the clinical interpretation of non-canonical splicing variants offers a major opportunity to uplift diagnostic yields from whole genome sequencing data. METHODS: Here, we examine the landscape of splicing variants in whole-genome sequencing data from 38,688 individuals in the 100,000 Genomes Project and assess the contribution of non-canonical splicing variants to rare genetic diseases. We use a variant-level constraint metric (the mutability-adjusted proportion of singletons) to identify constrained functional variant classes near exon-intron junctions and at putative splicing branchpoints. To identify new diagnoses for individuals with unsolved rare diseases in the 100,000 Genomes Project, we identified individuals with de novo single-nucleotide variants near exon-intron boundaries and at putative splicing branchpoints in known disease genes. We identified candidate diagnostic variants through manual phenotype matching and confirmed new molecular diagnoses through clinical variant interpretation and functional RNA studies. RESULTS: We show that near-splice positions and splicing branchpoints are highly constrained by purifying selection and harbour potentially damaging non-coding variants which are amenable to systematic analysis in sequencing data. From 258 de novo splicing variants in known rare disease genes, we identify 35 new likely diagnoses in probands with an unsolved rare disease. To date, we have confirmed a new diagnosis for six individuals, including four in whom RNA studies were performed. CONCLUSIONS: Overall, we demonstrate the clinical value of examining non-canonical splicing variants in individuals with unsolved rare diseases.

Germline de novo SETBP1 variants cause clinically distinct and heterogeneous neurodevelopmental disorders. Heterozygous missense variants at a hotspot encoding a canonical degron lead to SETBP1 accumulation and Schinzel-Giedion syndrome (SGS), a rare severe developmental disorder involving multisystem malformations. Heterozygous loss-of-function variants result in SETBP1 haploinsufficiency disorder which is phenotypically much milder than SGS. Following an initial description of four individuals with atypical SGS carrying heterozygous missense variants adjacent to the degron, a few individual cases of variants outside the degron were reported. Due to the lack of systematic investigation of genotype-phenotype associations of different types of SETBP1 variants, and limited understanding of the roles of the gene in brain development, the extent of clinical heterogeneity and how this relates to underlying pathophysiological mechanisms remain elusive, imposing challenges for diagnosis and patient care. Here, we present a comprehensive investigation of the largest cohort to-date of individuals carrying SETBP1 missense variants outside the degron (n=18, including one in-frame deletion). We performed thorough clinical and speech phenotyping with functional follow-up using cellular assays and transcriptomics. Our findings suggest that such variants cause a clinically and functionally variable developmental syndrome, showing only partial overlaps with classical SGS and SETBP1 haploinsufficiency disorder, and primarily characterised by intellectual disability, epilepsy, speech and motor impairment. We provide evidence of loss-of-function pathophysiological mechanisms impairing ubiquitination, DNA-binding and transcription. In contrast to SGS and SETBP1 haploinsufficiency, these effects are independent of protein abundance. Overall, our study provides important novel insights into diagnosis, patient care and aetiology of SETBP1-related disorders.

PurposeAlthough haploinsufficiency of ANKRD11 is among the most common genetic causes of neurodevelopmental disorders, the role of rare ANKRD11 missense variation remains unclear. We characterized the clinical, molecular and functional spectra of ANKRD11 missense variants.MethodsWe collected clinical information of individuals with ANKRD11 missense variants and evaluated phenotypic fit to KBG syndrome. We assessed pathogenicity of variants by in silico analyses and cell-based experiments.ResultsWe identified 29 individuals with (mostly de novo) ANKRD11 missense variants, who presented with syndromic neurodevelopmental disorders and were phenotypically similar to individuals with KBG syndrome caused by ANKRD11 protein truncating variants or 16q24.3 microdeletions. Missense variants significantly clustered in Repression Domain 2. Cellularly, most variants caused reduced ANKRD11 stability. One variant resulted in decreased proteasome degradation and loss of ANKRD11 transcriptional activity.ConclusionOur study indicates that pathogenic heterozygous missense variants in ANKRD11 cause the clinically recognizable KBG syndrome. Disrupted transrepression capacity and reduced protein stability each independently lead to ANKRD11 loss-of-function, consistent with haploinsufficiency. This highlights the diagnostic relevance of ANKRD11 missense variants, but also poses diagnostic challenges, as the KBG-associated phenotype may be mild and inherited pathogenic ANKRD11 (missense) variants are increasingly observed, warranting stringent variant classification and careful phenotyping.

Pathological cerebral white matter changes in Alzheimer's disease have been shown using diffusion tensor imaging. Superficial white matter changes are relatively understudied despite their importance in cortico-cortical connections. Measuring superficial white matter degeneration using diffusion tensor imaging is challenging due to its complex organizational structure and proximity to the cortex. To overcome this, we investigated diffusion MRI changes in young-onset Alzheimer's disease using standard diffusion tensor imaging and Neurite Orientation Dispersion and Density Imaging to distinguish between disease-related changes that are degenerative (e.g. loss of myelinated fibres) and organizational (e.g. increased fibre dispersion). Twenty-nine young-onset Alzheimer's disease patients and 22 healthy controls had both single-shell and multi-shell diffusion MRI. We calculated fractional anisotropy, mean diffusivity, neurite density index, orientation dispersion index and tissue fraction (1-free water fraction). Diffusion metrics were sampled in 15 a priori regions of interest at four points along the cortical profile: cortical grey matter, grey/white boundary, superficial white matter (1 mm below grey/white boundary) and superficial/deeper white matter (2 mm below grey/white boundary). To estimate cross-sectional group differences, we used average marginal effects from linear mixed effect models of participants' diffusion metrics along the cortical profile. The superficial white matter of young-onset Alzheimer's disease individuals had lower neurite density index compared to controls in five regions (superior and inferior parietal, precuneus, entorhinal and parahippocampus) (all P

Purpose: While behavioral aphasia therapy is beneficial (Brady et al., 2016), we do not fully understand factors that predict therapy response, or that contribute to extra-linguistic aspects of living with aphasia (e.g., psychosocial). The purpose of this Viewpoint is to postulate that inner speech – the ability to talk to oneself in one’s head – may be an important factor. However, prior work evaluating inner speech in aphasia has been limited in scope. Here, we innovatively draw from interdisciplinary evidence to discuss a more comprehensive view of inner speech and propose how evaluating a multidimensional inner speech may be meaningful in understanding living with aphasia and aphasia recovery. Methods: We give an interdisciplinary overview of inner speech, as it relates to aphasia. Results: Research with persons with aphasia shows that inner speech can be relatively spared in comparison to overt speech. However, this research has taken a narrow view of inner speech, defining inner speech as a covert ‘voice’ drawn upon during experimental tasks, such as object naming, rhyme decisions, or tongue twisters. Cross disciplinary research evaluating inner speech has identified its multidimensionality (specifically, dimensions of intentionality, condensation, and dialogality). Inner speech evaluated across these dimensions in neurotypical populations has shown that inner speech can be related to personal factors like self-awareness; retain phonetic features but also be like ‘thinking in pure pictures; and be both monologic and dialogic. Conclusions: Quantifying a multidimensional inner speech in aphasia will enable future work elaborating on factors related to extra-linguistic and linguistic processes of recovery, as well as living well with aphasia.

Alzheimer's disease (AD) is associated with extensive alterations in grey matter microstructure, but our ability to quantify this in vivo is limited. Neurite orientation dispersion and density imaging (NODDI) is a multi-shell diffusion MRI technique that estimates neuritic microstructure in the form of orientation dispersion and neurite density indices (ODI/NDI). Mean values for cortical thickness, ODI, and NDI were extracted from predefined regions of interest in the cortical grey matter of 38 patients with young onset AD and 22 healthy controls. Five cortical regions associated with early atrophy in AD (entorhinal cortex, inferior temporal gyrus, middle temporal gyrus, fusiform gyrus, and precuneus) and one region relatively spared from atrophy in AD (precentral gyrus) were investigated. ODI, NDI, and cortical thickness values were compared between controls and patients for each region, and their associations with MMSE score were assessed. NDI values of all regions were significantly lower in patients. Cortical thickness measurements were significantly lower in patients in regions associated with early atrophy in AD, but not in the precentral gyrus. Decreased ODI was evident in patients in the inferior and middle temporal gyri, fusiform gyrus, and precuneus. The majority of AD-related decreases in cortical ODI and NDI persisted following adjustment for cortical thickness, as well as each other. There was evidence in the patient group that cortical NDI was associated with MMSE performance. These data suggest distinct differences in cortical NDI and ODI occur in AD and these metrics provide pathologically relevant information beyond that of cortical thinning.

Objective To compare the economic costs between two strategies for management of the small unruptured ectopic pregnancy: initial treatment with methotrexate versus initial treatment with laparoscopic salpingostomy. Methods We assumed that both treatment strategies would result in identical clinical outcomes: resolution of the ectopic pregnancy without maternal mortality or long-term morbidity. Based on a literature review, estimates were derived for the likely clinical outcomes of a single injection of methotrexate (50 mg/m2) and for the likely clinical outcomes of the laparoscopy strategy. A range of values was evaluated for the initial success rate of each strategy and varying assumptions made about the type of treatment modality used for initial treatment failures. Direct medical costs of each strategy were estimated based on actual reimbursement rates of a third-party payer for the components of each strategy. The treatment strategies were compared in best-case/worst-case scenarios to determine the potential range of differences in costs between the two strategies. Results The cost of the methotrexate strategy ranged from $438 to $1390, and the cost of laparoscopic salpingostomy ranged from $2506 to $2974; therefore, the methotrexate strategy was less costly than laparoscopy, with a cost difference ranging from $1124 (best-case laparoscopy-worst-case methotrexate scenario) to $2536 (worst-case laparoscopybest-case methotrexate scenario). Sensitivity analyses demonstrated that initial therapy with methotrexate was less costly over a wide range of probability and cost estimates. Conclusion Initial methotrexate is a cost-effective alternative to laparoscopic salpingostomy in the treatment of the small unruptured ectopic pregnancy.