Clinical findings and structural analysis involving a patient with a novel KLHL15 variant.

A de novo novel variant of uncertain significance p. (Arg532del) in the KLHL15 gene was identified by trio exome analysis in a child with global developmental delay, coarse facial features, repetitive behaviour, increased fatigability, poor feeding and gastro-oesophageal reflux. Comparative modelling and structural analysis were performed to gain insight into the effects of the variant on KLHL15 protein structure and function, with a view to aiding variant classification. The p. (Arg532del) variant affects a highly conserved residue within one of the Kelch repeats of the KLHL15 protein. This residue contributes to the stability of loop regions at the substrate binding surface of the protein; comparative modelling of the variant protein predicts altered topology at this surface, including at residue Tyr552, which is known to be important for substrate binding. We propose that it is highly probable that the p. (Arg532del) variant has a deleterious impact on KLHL15 structure, leading to a reduced level of protein function in vivo.

Combination Therapies Targeting Apoptosis in Paediatric AML: Understanding the Molecular Mechanisms of AML Treatments Using Phosphoproteomics.

Paediatric acute myeloid leukaemia (AML) continues to present treatment challenges, as no "standard approach" exists to treat those young patients reliably and safely. Combination therapies could become a viable treatment option for treating young patients with AML, allowing multiple pathways to be targeted. Our in silico analysis of AML patients highlighted "cell death and survival" as an aberrant, potentially targetable pathway in paediatric AML patients. Therefore, we aimed to identify novel combination therapies to target apoptosis. Our apoptotic drug screening resulted in the identification of one potential "novel" drug pairing, comprising the Bcl-2 inhibitor ABT-737 combined with the CDK inhibitor Purvalanol-A, as well as one triple combination of ABT-737 + AKT inhibitor + SU9516, which showed significant synergism in a series of paediatric AML cell lines. Using a phosphoproteomic approach to understand the apoptotic mechanism involved, proteins related to apoptotic cell death and cell survival were represented, in agreement with further results showing differentially expressed apoptotic proteins and their phosphorylated forms among combination treatments compared to single-agent treated cells such upregulation of BAX and its phosphorylated form (Thr167), dephosphorylation of BAD (Ser 112), and downregulation of MCL-1 and its phosphorylated form (Ser159/Thr 163). Total levels of Bcl-2 were decreased but correlated with increased levels of phosphorylated Bcl-2, which was consistent with our phosphoproteomic analysis predictions. Bcl-2 phosphorylation was regulated by extracellular-signal-regulated kinase (ERK) but not PP2A phosphatase. Although the mechanism linking to Bcl-2 phosphorylation remains to be determined, our findings provide first-hand insights on potential novel combination treatments for AML.

Genetic testing in motor neuron disease and frontotemporal dementia: a 5-year multicentre evaluation.

INTRODUCTION: Motor neuron disease (MND) and frontotemporal dementia (FTD) comprise a neurodegenerative disease spectrum. Genetic testing and counselling is complex in MND/FTD owing to incomplete penetrance, variable phenotype and variants of uncertain significance. Affected patients and unaffected relatives are commonly referred to clinical genetics to consider genetic testing. However, no consensus exists regarding how such genetic testing should best be undertaken and on which patients. OBJECTIVE: We sought to ascertain UK clinical genetics testing practice in MND/FTD referrals, with the aim of helping inform guideline development. METHODS: MND/FTD clinical genetics referrals comprising both affected patients and unaffected relatives between 2012 and 2016 were identified and a standardised proforma used to collate data from clinical records. RESULTS: 301 referrals (70 affected, 231 unaffected) were reviewed across 10 genetics centres. Previously identified familial variants were known in 107 cases and 58% subsequently underwent testing (8 of 8 diagnostic and 54 of 99 predictive). The median number of genetic counselling appointments was 2 for diagnostic and 4 for predictive testing. Importantly, application of current UK Genomic Test Directory eligibility criteria would not have resulted in detection of all pathogenic variants observed in this cohort. CONCLUSION: We propose pragmatic MND/FTD genetic testing guidelines based on appropriate genetic counselling.

Multiplex Screening for Interacting Compounds in Paediatric Acute Myeloid Leukaemia.

Paediatric acute myeloid leukaemia (AML) is a heterogeneous disease characterised by the malignant transformation of myeloid precursor cells with impaired differentiation. Standard therapy for paediatric AML has remained largely unchanged for over four decades and, combined with inadequate understanding of the biology of paediatric AML, has limited the progress of targeted therapies in this cohort. In recent years, the search for novel targets for the treatment of paediatric AML has accelerated in parallel with advanced genomic technologies which explore the mutational and transcriptional landscape of this disease. Exploiting the large combinatorial space of existing drugs provides an untapped resource for the identification of potential combination therapies for the treatment of paediatric AML. We have previously designed a multiplex screening strategy known as Multiplex Screening for Interacting Compounds in AML (MuSICAL); using an algorithm designed in-house, we screened all pairings of 384 FDA-approved compounds in less than 4000 wells by pooling drugs into 10 compounds per well. This approach maximised the probability of identifying new compound combinations with therapeutic potential while minimising cost, replication and redundancy. This screening strategy identified the triple combination of glimepiride, a sulfonylurea; pancuronium dibromide, a neuromuscular blocking agent; and vinblastine sulfate, a vinca alkaloid, as a potential therapy for paediatric AML. We envision that this approach can be used for a variety of disease-relevant screens allowing the efficient repurposing of drugs that can be rapidly moved into the clinic.

COVID-19 myopericarditis with cardiac tamponade in the absence of respiratory symptoms: a case report.

BACKGROUND: Previous reports have shown various cardiac complications to be associated with COVID-19 including: myocardial infarction, microembolic complications, myocardial injury, arrythmia, heart failure, coronary vasospasm, non-ischemic cardiomyopathy, stress (Takotsubo) cardiomyopathy, pericarditis and myocarditis. These COVID-19 cardiac complications were associated with respiratory symptoms. However, our case illustrates that COVID-19 myopericarditis with cardiac tamponade can present without respiratory symptoms. CASE PRESENTATION: A 58-year-old Caucasian British woman was admitted with fever, diarrhoea and vomiting. She developed cardiogenic shock and Transthoracic echocardiogram (TTE) found a pericardial effusion with evidence of cardiac tamponade. A nasopharyngeal swab showed a COVID-19 positive result, despite no respiratory symptoms on presentation. A pericardial drain was inserted and vasopressor support required on intensive treatment unit (ITU). The drain was removed as she improved, an antibiotic course was given and she was discharged on day 12. CONCLUSIONS: Our case demonstrates that patients without respiratory symptoms could have COVID-19 and develop cardiac complications. These findings can aid timely diagnosis of potentially life-threatening COVID-19 myopericarditis with cardiac tamponade.

Uncoupled turnover disrupts mitochondrial quality control in diabetic retinopathy.

Mitochondrial quality control (MQC) is crucial for regulating CNS homeostasis, and its disruption has been implicated in the pathogenesis of some of the most common neurodegenerative diseases. In healthy tissues, the maintenance of MQC depends upon an exquisite balance between mitophagy (removal of damaged mitochondria by autophagy) and biogenesis (de novo synthesis of mitochondria). Here, we show that mitophagy is disrupted in diabetic retinopathy (DR) and decoupled from mitochondrial biogenesis during the progression of the disease. Diabetic retinas from human postmortem donors and experimental mice exhibit a net loss of mitochondrial contents during the early stages of the disease process. Using diabetic mitophagy-reporter mice (mitoQC-Ins2Akita) alongside pMitoTimer (a molecular clock to address mitochondrial age dynamics), we demonstrate that mitochondrial loss arose due to an inability of mitochondrial biogenesis to compensate for diabetes-exacerbated mitophagy. However, as diabetes duration increases, Pink1-dependent mitophagy deteriorates, leading to the build-up of mitochondria primed for degradation in DR. Impairment of mitophagy during prolonged diabetes is linked with the development of retinal senescence, a phenotype that blunted hyperglycemia-induced mitophagy in mitoQC primary Müller cells. Our findings suggest that normalizing mitochondrial turnover may preserve MQC and provide therapeutic options for the management of DR-associated complications.

Glucose transporter 1 expression as a marker of prognosis in oesophageal adenocarcinoma.

BACKGROUND: The current TNM staging system for oesophageal adenocarcinoma (OAC) has limited ability to stratify patients and inform clinical management following neo-adjuvant chemotherapy and surgery. RESULTS: Functional genomic analysis of the gene expression data using Gene Set Enrichment Analysis (GSEA) identified GLUT1 as putative prognostic marker in OAC.In the discovery cohort GLUT1 positivity was observed in 114 patients (80.9%) and was associated with poor overall survival (HR 2.08, 95% CI 1.1-3.94; p=0.024) following multivariate analysis. A prognostic model incorporating GLUT1, CRM and nodal status stratified patients into good, intermediate and poor prognosis groups (p< 0.001) with a median overall survival of 16.6 months in the poorest group.In the validation set 182 patients (69.5%) were GLUT1 positive and the prognostic model separated patients treated with neo-adjuvant chemotherapy and surgery (p<0.001) and surgery alone (p<0.001) into three prognostic groups. PATIENTS AND METHODS: Transcriptional profiling of 60 formalin fixed paraffin-embedded (FFPE) biopsies was performed. GLUT1 immunohistochemical staining was assessed in a discovery cohort of 141 FFPE OAC samples treated with neo-adjuvant chemotherapy and surgery at the Northern Ireland Cancer Centre from 2004-2012. Validation was performed in 262 oesophageal adenocarcinomas collected at four OCCAMS consortium centres. The relationship between GLUT1 staining, T stage, N stage, lymphovascular invasion and circumferential resection margin (CRM) status was assessed and a prognostic model developed using Cox Proportional Hazards. CONCLUSIONS: GLUT1 staining combined with CRM and nodal status identifies a poor prognosis sub-group of OAC patients and is a novel prognostic marker following potentially curative surgical resection.