Whitchurch Hospital

Systematic analysis of copy number variants (CNVs) in large datasets is challenging and there are limited studies of homozygous copy number losses in rare disease exome datasets. Here we leveraged the genomic uniqueness and relative under-representation of the Indian population in the current public genomic databases and identified 42,386 possible homozygous losses (median count 20 per individual, range 0 - 55; median size 2.95 kb, range 99 bp - 4.76 Mb) in a heterogeneous cohort of 2,021 individuals with suspected Mendelian disorders, who had undergone exome sequencing using 12 different capture kits in a resource-limited setting. Employing a genomic position loss-count based approach, we filtered 1,224 rare homozygous loss calls in 718 individuals (median count 1 per individual, range 0 - 22; median size 3.49 kb, range 121 bp - 4.76 Mb) for further analysis, thus significantly reducing the analysis burden. Clinical correlation and validation of these rare calls enabled 10 new diagnoses in 240 unsolved individuals with at least one filtered rare homozygous loss call. This, led to nearly two-fold increase in diagnosis owing to homozygous deletions in our cohort. Further analysis of the data and identification of additional affected individuals through collaboration led to identification of biallelic FILIP1 and FAM177A1 variants as causes of a syndromic arthrogryposis and a neuromuscular disorder respectively. Both these conditions have been recently proven as ultra-rare recessive disorders, thus validating our approach. We also show that biallelic loss-of-function TFCP2L1 variants cause chronic kidney disease and VPS36 variants cause a severe recessive neurodevelopmental disorder characterised by microcephaly, motor delay, agenesis of the corpus callosum, cerebellar atrophy, seizures, hypotonia, spasticity and early death. Overall, these results demonstrate a scalable approach to screen homozygous losses for improving diagnostic yield and discovering disease-genes in large exome cohorts.

Abstract Background Targeting melanoma by T cells drives anti-tumor responses. We previously showed that a DNA vaccine, SCIB1, incorporating T cell epitopes from TRP-2/gp100 into an antibody framework to allow Fc targeting of activated dendritic cells, was successfully evaluated as a monotherapy in a phase 1/2 in stage 3/4 melanoma patients. Unresectable melanoma patients showed a 60% ORR and 88% of patients treated with SCIB1 post tumor resection, remained disease free for 5 years [1]. The current SCOPE trial tests the hypothesis that unresectable patients may have an improved response when SCIB1 is combined with checkpoint inhibitors (CPI). Methods Patients were treated with nivolumab with ipilimumab and SCIB1 (8mg) i.m. using needle-free injection at a fixed dosing schedule for a total of 10 doses over 24 months. The CPI therapy was administered i.v. in accordance with their respective SmPC. ORR as measured by RECIST 1.1 in the overall intention-to-treat population was the primary endpoint. The study is designed using Simon's two stage methodology with 80% power when the true response rate is 70% with an overall type I error of 5%. In the first stage, 15 patients will be enrolled and if there are eight or fewer clinical responses (RECIST 1.1 objective response [CR or PR] within 25 weeks of the first dose of SCIB1), further recruitment will be stopped. The null hypothesis will be rejected if 27 or more responses are observed in 43 patients. Results 19 patients received the combination of SCIB1 with nivolumab and ipilimumab. At study entry, all patients were stage IV. 13 patients had reached the first imaging timepoint at 13 weeks, and the objective response rate is 85%. 9/9 responses were confirmed in a subsequent scan. Patients showed a 40-95% reduction in tumor volume between 13 and 25 weeks. Most of the SCIB1-related adverse events were Grade 1/2. Only 1 patient reported a Grade 3 rash. No enhancement of immune-mediated adverse events was observed when SCIB1 was added to nivolumab with ipilimumab. Conclusions SCIB1 in combination with nivolumab and ipilimumab as first line treatment for unresectable melanoma improved the ORR to 85% without an increase in clinically meaningful adverse events. These results if confirmed in a larger patient cohort provide confidence in initiating a randomized registration program in unresectable melanoma patients with the novel DNA plasmid technology Citation Format: Heather Shaw, Poulam Patel, Miranda Payne, Satish Kumar, Martin Highley, Kellati Prasad, Ruth Board, Clare Barlow, Sarah Danson, Robert Miller, Georgia Goodhew, Fayaz Master, Lindy Durrant. A DNA plasmid melanoma cancer vaccine, SCIB1, combined with nivolumab + ipilimumab in patients with advanced unresectable melanoma: Efficacy and safety results from the open-label Phase 2 SCOPE Trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT024.

Abstract Background. Malignant melanoma exhibits distinct lipid profiles compared to normal tissue and changes in the lipidome influence multiple aspects of tumor biology. Lipidomic alterations within the tumor microenvironment (TME) may influence the efficacy of anti-PD-1 therapy. In combination with anti-PD-1 agent pembrolizumab, NUC-7738 (3'-deoxyadenosine [3’-dA] which is phosphorylated and protected with a phosphoramidate moiety attached at the 5’-position) has shown promising activity in anti-PD-1 refractory melanoma (Phase 2; NCT03829254). NUC-7738 generates sustained intracellular levels of 3’-dATP, which profoundly alters RNA regulatory processes in tumor cells, resulting in impaired cellular responses and changes in expression of proteins related to lipid biosynthesis. To interrogate components of the TME before and after NUC-7738 + pembrolizumab treatment, we developed a novel workflow incorporating Mass Spectrometry Imaging (MSI) to co-register lipidomic changes with molecular markers. Machine learning accelerated the interpretation of complex data obtained from paired patient biopsies. Methods. Snap frozen paired biopsies from patients with advanced cutaneous melanoma treated with NUC-7738 ±pembrolizumab were analyzed by desorption electrospray ionization (DESI)-MSI. Specific metabolic (e.g., ASAH1) and immune (e.g., CD8, CD20) markers were investigated by multiplex immunofluorescence (mIF) post DESI-MSI. Target selection was guided by bulk RNA-seq data. Raw MSI data was visualized using Waters HDI® software. Data extracted from selected regions of interest was exported into MetaboAnalyst 5.0 for statistical analysis. LIPID MAPS® Structure Database was used to assign lipid species. mIF/mIHC images were analyzed by HALO® AI. Results. Single 10 µm tissue sections allowed co-registered DESI-MSI and mIF, suitable for further informatic and AI-enabled image analysis. This facilitated direct comparison of lipids, cells, and protein expression in the TME, before and after treatment with NUC-7738, a compound known to cause profound changes in poly(A) tail regulation. MSI data analyses of the TME uncovered intra-tumoral heterogeneity of the lipidome and demonstrated changes following treatment. Conclusion. This platform provides a powerful investigative tool to simultaneously explore the myriad of factors in the TME that contribute to tumorigenesis and therapeutic response. Further analyses of the role of the lipidome in the TME and intra-tumoral heterogeneity will be explored in paired biopsies from patients with PD-1 inhibitor refractory melanoma. Citation Format: Alison L. Dickson, Greice M. Zickuhr, In Hwa Um, Ying Zhang, Ruth Plummer, Sarah P. Blagden, Stefan N. Symeonides, Natalie Cook, T.R. Jeffry Evans, Mustafa Elshani, David J. Harrison. Exposing the heterogeneity of the lipidome in the TME of cutaneous melanoma following treatment with NUC-7738 in combination with anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6222.

Joint pain makes up a significant proportion of presentations to general practice. Causes range from minor mechanical issues to serious conditions such as septic arthritis. Clues from the history such as onset, progression, and extra-articular symptoms aid diagnosis. Many cases of joint pain are appropriately investigated and managed in general practice, but some presentations require referral to secondary care, such as those with signs or symptoms of inflammatory arthritis. It is crucial that red flags for serious or life-threatening causes of joint pain are identified and acted upon urgently.

ABSTRACT Large-scale intervention in the Earth’s climate system is increasingly present in discussions about possible responses to climate change. Young people’s perspectives have tended to be under-represented despite the intergenerational consequences of policy in this field. We report on a novel approach to research and practice: the co-creation of a youth guide and policy brief by youth participants and facilitators. The model offers potential use by practitioners for engaging publics at the early stages of technoscientific innovations. Findings fall into two categories: youth priorities for geoengineering and authorial responsibility as a way of supporting youth action. Tentative conclusions from youth participants are (i) action must be prioritised now to mitigate and adapt to climate change, rather than continuing with ‘business as usual’; and (ii) there is a need for proactive international cooperation on governance and research on geoengineering to understand potential environmental and social consequences of geoengineering proposals for people at different temporal and spatial scales. Greater public dialogue on geoengineering and its governance is needed, particularly involving young people. The youth guide and policy brief co-authored by participants and facilitators, and the dialogic methods used in their production, can contribute to this dialogue.

Veterinary anaesthesia may be facing new challenges during the COVID-19 pandemic, with the potential for shortages of cylinders of oxygen and some drugs in veterinary practice if resources are diverted to the NHS. This article prepares the veterinary surgeon in the event of such shortages, and discusses how to safely limit the use of oxygen in practice as well as the use of alfaxalone as an alternative induction and maintenance agent to propofol. Finally, the use of ephedrine to manage hypotension as an alternative to other vasopressors and inotropes that may be in short supply is described.

Interactions between iron surfaces and hydrocarbons are the basis for a wide range of materials synthesis processes and novel applications, including sensing. However, in diesel engines these interactions can lead to deposit formation that reduces performance, lowers efficiency, and increases emissions. Here, we present a global study to understand deposition at iron-hexadecane interfaces. We use a combination of spectroscopy, microscopy, and mass spectrometry to investigate surface reactions, bulk chemistry, and deposition processes. A dynamic equilibrium between the oxidation products, both at the surface and in solution, determines the deposition at the surface. Considering the solution and the surface in parallel, we find that the iron speciation affects the morphology, composition, and quantity of the deposit at the surface, as well as the oxidation of hexadecane. Fe(II) and Fe(III) both promote the decomposition of peroxides-intermediates in the oxidation of hexadecane-but through noncatalytic and catalytic mechanisms, respectively. In contrast, Fe(0) is proposed to initiate hexadecane autoxidation during its oxidation to Fe(III). We find that in all cases, the surfaces exclusively contain Fe(III) following heat treatment with hexadecane. Upon subsequent exposure at room temperature, Fe(III) species are found to promote oxidation; this finding is particularly concerning for hybrid vehicles where longer time periods are expected between engine operation. Our work provides a foundation for the development of strategies that disrupt the role of iron in the degradation of hexadecane to ultimately reduce oxidation and deposition in diesel engines.

To develop and validate a self-report measure designed to assess perceived and experienced stigma for adults with Type 1 diabetes: the Type 1 Diabetes Stigma Assessment Scale (DSAS-1). A large item-pool (64 items) was drafted based on qualitative data from interviews with 27 adults with Type 1 diabetes. Eleven adults with Type 1 diabetes completed the draft questionnaire (responding to items using a five-point Likert scale), and participated in cognitive debriefing interviews. Based on their feedback, the item-pool was reduced and refined. Adults with Type 1 diabetes (N=898) completed an online survey including the draft stigma questionnaire (41 items) and other validated measures. Psychometric validation included principal components analysis and confirmatory factor analysis (split samples), internal consistency reliability assessment and Spearman's rho correlations. Scale reduction techniques resulted in 19 items (α=0.93). An unforced three-factor solution suggested three subscales: Treated Differently (six items, α=0.89); Blame and Judgement (six items, α=0.88); and Identity Concerns (seven items, α=0.89). This was corroborated with a confirmatory factor analysis, which demonstrated reasonable model fit with the three factors; less so for a single-factor model. Satisfactory concurrent, convergent and discriminant validity were demonstrated. The 19-item DSAS-1 is a valid and reliable measure of the perceptions and experiences of Type 1 diabetes stigma. This novel, relatively brief measure has satisfactory psychometric properties. The DSAS-1 is now available for investigations into the nature and magnitude of the relationships between diabetes stigma and diabetes self-care behaviours and outcomes.

ObjectiveTo provide preliminary evidence for the impact of problem-solving therapy for diabetes (PST-D) in adults with diabetic retinopathy (DR) and diabetes distress.Research design and methodsIn a pilot randomized controlled trial, 40 participants with DR and diabetes distress were allocated to the PST-D or control groups. Diabetes distress (DDS), depressive symptoms (PHQ-9), self-care activities (SDSCA), and HbA1c were assessed at baseline, and 3 and 6-month follow-ups.ResultsAt the 6-month follow-up, the PST-D group showed significant improvements relative to the control group, in ‘regimen-related distress’ (PST-D: −1.3±1.4; control: −0.4±1.1), depressive symptoms (PST-D: −4.3±6.1; control: −0.3±4.6), and HbA1c (PST-D: −1.2%±1.01; control: 0.2%±1.2%) (all p<0.05). In multiple regression analysis, adjusting for baseline values and sociodemographic factors, PST-D was associated with significant improvement in ‘regimen-related distress’, depressive symptoms, and HbA1c at the 6-month follow-up (p<0.05).ConclusionsPST-D is a promising intervention for improving psychological outcomes and glycemic control. A fully powered study is required to confirm these findings and examine mechanisms of change in HbA1c.Trial registration numberACTRN12616001010482; results.

ABSTRACTAutosomal dominant hypocalcemia (ADH) is characterized by hypocalcemia, inappropriately low serum parathyroid hormone concentrations and hypercalciuria. ADH is genetically heterogeneous with ADH type 1 (ADH1), the predominant form, being caused by germline gain‐of‐function mutations of the G‐protein coupled calcium‐sensing receptor (CaSR), and ADH2 caused by germline gain‐of‐function mutations of G‐protein subunit α‐11 (Gα11). To date Gα11 mutations causing ADH2 have been reported in only five probands. We investigated a multigenerational nonconsanguineous family, from Iran, with ADH and keratoconus which are not known to be associated, for causative mutations by whole‐exome sequencing in two individuals with hypoparathyroidism, of whom one also had keratoconus, followed by cosegregation analysis of variants. This identified a novel heterozygous germline Val340Met Gα11 mutation in both individuals, and this was also present in the other two relatives with hypocalcemia that were tested. Three‐dimensional modeling revealed the Val340Met mutation to likely alter the conformation of the C‐terminal α5 helix, which may affect G‐protein coupled receptor binding and G‐protein activation. In vitro functional expression of wild‐type (Val340) and mutant (Met340) Gα11 proteins in HEK293 cells stably expressing the CaSR, demonstrated that the intracellular calcium responses following stimulation with extracellular calcium, of the mutant Met340 Gα11 led to a leftward shift of the concentration‐response curve with a significantly (p < 0.0001) reduced mean half‐maximal concentration (EC50) value of 2.44 mM (95% CI, 2.31 to 2.77 mM) when compared to the wild‐type EC50 of 3.14 mM (95% CI, 3.03 to 3.26 mM), consistent with a gain‐of‐function mutation. A novel His403Gln variant in transforming growth factor, beta‐induced (TGFBI), that may be causing keratoconus was also identified, indicating likely digenic inheritance of keratoconus and ADH2 in this family. In conclusion, our identification of a novel germline gain‐of‐function Gα11 mutation, Val340Met, causing ADH2 demonstrates the importance of the Gα11 C‐terminal region for G‐protein function and CaSR signal transduction. © 2016 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).