Understanding the Cellular Consequences of Meier-Gorlin Syndrome

Meier-Gorlin syndrome (MGS) is a rare autosomal recessive disorder characterised by short stature, microtia and patella aplasia/hypoplasia. Genetic variants, which cause MGS, have previously been found in genes involved in the initiation of DNA replication. This study examined the cellular consequences of novel variants in MGS genes; ORC1, CDC45 and DONSON. The ORC1 gene encodes an essential component of the pre-replication complex and functions during late mitosis/early G1 phase to initiate DNA replication. MGS individuals previously reported to have variants in ORC1 have had at least one variant in the bromo-adjacent homology (BAH) domain at the N-terminus of the protein, a region suggested to be important for protein-protein interactions. In this study we report a patient with a novel homozygous variant (c.1865T>C, p.L622P) in the ATPase Associated with a wide range of cellular Activities (AAA) domain at the C-terminus of ORC1, and attempted to investigate how variants in this region of the protein lead to a MGS phenotype using CRISPR-Cas9 genome editing and a minigene splicing assay. Due to time constraints, results were inconclusive and more work is required to better understand variants in this area of ORC1. CDC45 encodes an essential component of the pre-initiation and CDC45-MCM-GINS (CMG) complex, required during G1 and S-phase. The novel homozygous CDC45 variant (c. 1441-2 A>G) under investigation in this study was found in two siblings presenting with a severe MGS phenotype alongside a range of secondary phenotypes some of which are not typically associated with MGS. It was hypothesised that this novel variant may represent the extreme end of the CDC45 phenotypic spectrum. A splicing assay showed that the variant, located within the canonical splice acceptor site for CDC45 exon 16, caused aberrant splicing and use of an alternative 5′ splice acceptor within exon 16. This resulted in a two amino acid deletion (p.Thr481_Lys482del) and a 72.9% reduction in CDC45 mRNA levels, which was confirmed by RT-qPCR analysis using patient fibroblasts. DONSON is required for stabilising replication forks during S-phase when replication stress is encountered. Biallelic variants in this gene have previously been described in patients presenting with Microcephaly-Micromelia syndrome (MIMIS) and Microcephaly Short Stature, and Limb Abnormalities (MISSLA), both of which are characterized by severe microcephaly and a slight reduction in height. In this study we discovered a novel DONSON variant (c.631C>T, p.R211C), for the first time in clinically diagnosed MGS patients, presenting with a global reduction in size. This project used CRISPR-Cas9 genome editing as well as a range of cellular techniques including subcellular localisation, immunocytochemistry and DNA fibre combing to better understand the cellular consequences of this novel MGS gene. Taken together, results confirmed DONSON as a novel MGS gene and showed that MGS variants led to more subtle changes in subcellular localisation, DNA damage, and replication events than variants seen in non-MGS DONSON patients, reflecting the difference in phenotype. This project aimed to study the cellular consequences of novel MGS variants using CRISPR-Cas9 and a variety of cellular techniques to understand the effect these novel variants had on initiation of DNA replication

An eHealth Program for patients undergoing a total hip arthroplasty: Protocol for a randomized controlled trial

Background: Total hip arthroplasty is an effective surgical procedure commonly used worldwide for patients suffering the disabling effects of osteoarthritis when medical therapy is unsuccessful. Traditionally pre- and postoperative information for patients undergoing a hip arthroplasty has been provided by paper-based methods. Electronic health (eHealth) programs to support individualized patient education on preoperative preparation, in-patient care, and home rehabilitation have the potential to increase patient engagement, enhance patient recovery, and reduce potential postoperative complications. Objective: The aim of this study is to compare the addition of an eHealth program versus standard care for pre- and postoperative education on patient outcomes for primary total hip arthroplasty. Methods: One hundred patients undergoing a primary elective total hip arthroplasty will be recruited from a metropolitan hospital in Western Australia to participate in a 6-month parallel randomized control trial. Participants will be randomized to either the standard care group (n=50) and will be given the education booklet and enrolled to attend a 1-hour education session, or the intervention group (n=50), and will receive the same as the standard care plus access to an eHealth program titled “My Hip Journey.” The eHealth program encourages the patient to log in daily, from 2 weeks prior to surgery to 30 days postsurgery. The information on the platform will be aligned with the patient\u27s individual surgical journey and will include exercises to be completed each day for the duration of the program. The primary outcome measure is the Hip Dysfunction and Osteoarthritis Outcome Score, version LK 2.0. Secondary outcome measures include the EuroQoL EQ-5D-5L, a 5-level 5-dimension quality of life measure, and the Self-Efficacy for Managing Chronic Disease Scale. Data will be collected at pre-admission (presurgery) and at 6 weeks, 3 months, and 6 months postsurgery. A patient satisfaction survey will be completed 6 weeks postsurgery and Web-based analytics will be collected 6 months postsurgery. A cost-effectiveness analysis, using the intention-to-treat principle, will be conducted from the hospital’s perspective. Results: Enrollment in the study commenced in January 2018 with recruitment due for completion towards the end of the year. The first results are expected to be submitted for publication in 2019. Conclusions: The outcomes and cost of using an eHealth program to support a patient’s recovery from a hip arthroplasty will be compared with standard care in this study. If the eHealth program is found to be effective, further implementation across clinical practice could lead to improvement in patient outcomes and other surgical areas could be incorporated

Malaria and pregnancy: placental cytokine expression and its relationship to intrauterine growth retardation

Malaria infections during pregnancy can lead to the delivery of low-birth-weight infants. In this study, cytokine mRNA was measured in placentas from 23 malaria-infected and 21 uninfected primigravid women who had delivered in Mangochi, Malawi, a region with a high rate of transmission of falciparum malaria. Significantly increased expression of interleukin (IL)-1beta, IL-8, and tumor necrosis factor (TNF)-alpha and decreased expression of IL-6 and transforming growth factor-beta1 were found in malaria-infected compared with uninfected placentas. TNF-alpha and IL-8 were produced by maternally derived hemozoin-laden placental macrophages. Increased TNF-alpha expression was associated with increased placental hemozoin concentrations. Increased TNF-alpha or IL-8 expression in the placenta was associated with intrauterine growth retardation but not with preterm delivery. The results suggest that malaria infections induce a potentially harmful proinflammatory response in the placenta

Neuroimaging markers for studying Gulf-War illness: single-subject level analytical method based on machine learning

Gulf War illness (GWI) refers to the multitude of chronic health symptoms, spanning from fatigue, musculoskeletal pain, and neurological complaints to respiratory, gastrointestinal, and dermatologic symptoms experienced by about 250,000 GW veterans who served in the 1991 Gulf War (GW). Longitudinal studies showed that the severity of these symptoms often remain unchanged even years after the GW, and these veterans with GWI continue to have poorer general health and increased chronic medical conditions than their non-deployed counterparts. For better management and treatment of this condition, there is an urgent need for developing objective biomarkers that can help with simple and accurate diagnosis of GWI. In this study, we applied multiple neuroimaging techniques, including T1-weighted magnetic resonance imaging (T1W-MRI), diffusion tensor imaging (DTI), and novel neurite density imaging (NDI) to perform both a group-level statistical comparison and a single-subject level machine learning (ML) analysis to identify diagnostic imaging features of GWI. Our results supported NDI as the most sensitive in defining GWI characteristics. In particular, our classifier trained with white matter NDI features achieved an accuracy of 90% and F-score of 0.941 for classifying GWI cases from controls after the cross-validation. These results are consistent with our previous study which suggests that NDI measures are sensitive to the microstructural and macrostructural changes in the brain of veterans with GWI, which can be valuable for designing better diagnosis method and treatment efficacy studies.W81XWH-17-1-0440 - a department of Defense CDMRP new investigator awardPublished versio

North Carolina public school teachers’ contact patterns and mask use within and outside of school during the pre-vaccine phase of the COVID-19 pandemic

Background : Teachers are central to school-associated transmission networks, but little is known about their behavioral patterns during the COVID-19 pandemic. Methods : We conducted a cross-sectional survey of 700 North Carolina public school teachers in four districts open to in-person learning in November-December 2020 (pre-COVID-19 vaccines). We assessed indoor and outdoor time spent, numbers of people encountered at 94%) reported wearing masks inside school, stores, and salons; intermediate percentages (∼50%-85%) inside places of worship, bars/restaurants, and recreational settings; and few (<25%) in their or others’ homes. Approximately half reported daily close contact with students. Conclusions : As schools reopened in the COVID-19 pandemic, potential transmission opportunities arose through close contacts within and outside of school, along with suboptimal mask use by teachers and/or those around them. Our granular estimates underscore the importance of multi-layered mitigation strategies and can inform interventions and mathematical models addressing school-associated transmission

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Integrated genomic characterization of pancreatic ductal adenocarcinoma

We performed integrated genomic, transcriptomic, and proteomic profiling of 150 pancreatic ductal adenocarcinoma (PDAC) specimens, including samples with characteristic low neoplastic cellularity. Deep whole-exome sequencing revealed recurrent somatic mutations in KRAS, TP53, CDKN2A, SMAD4, RNF43, ARID1A, TGFβR2, GNAS, RREB1, and PBRM1. KRAS wild-type tumors harbored alterations in other oncogenic drivers, including GNAS, BRAF, CTNNB1, and additional RAS pathway genes. A subset of tumors harbored multiple KRAS mutations, with some showing evidence of biallelic mutations. Protein profiling identified a favorable prognosis subset with low epithelial-mesenchymal transition and high MTOR pathway scores. Associations of non-coding RNAs with tumor-specific mRNA subtypes were also identified. Our integrated multi-platform analysis reveals a complex molecular landscape of PDAC and provides a roadmap for precision medicine

Effects of antiplatelet therapy after stroke due to intracerebral haemorrhage (RESTART): a randomised, open-label trial

Background: Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods: The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings: Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation: These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention