Evaluation of the feasibility, diagnostic yield, and clinical utility of rapid genome sequencing in infantile epilepsy (Gene-STEPS): an international, multicentre, pilot cohort study

BACKGROUND: Most neonatal and infantile-onset epilepsies have presumed genetic aetiologies, and early genetic diagnoses have the potential to inform clinical management and improve outcomes. We therefore aimed to determine the feasibility, diagnostic yield, and clinical utility of rapid genome sequencing in this population. METHODS: We conducted an international, multicentre, cohort study (Gene-STEPS), which is a pilot study of the International Precision Child Health Partnership (IPCHiP). IPCHiP is a consortium of four paediatric centres with tertiary-level subspecialty services in Australia, Canada, the UK, and the USA. We recruited infants with new-onset epilepsy or complex febrile seizures from IPCHiP centres, who were younger than 12 months at seizure onset. We excluded infants with simple febrile seizures, acute provoked seizures, known acquired cause, or known genetic cause. Blood samples were collected from probands and available biological parents. Clinical data were collected from medical records, treating clinicians, and parents. Trio genome sequencing was done when both parents were available, and duo or singleton genome sequencing was done when one or neither parent was available. Site-specific protocols were used for DNA extraction and library preparation. Rapid genome sequencing and analysis was done at clinically accredited laboratories, and results were returned to families. We analysed summary statistics for cohort demographic and clinical characteristics and the timing, diagnostic yield, and clinical impact of rapid genome sequencing. FINDINGS: Between Sept 1, 2021, and Aug 31, 2022, we enrolled 100 infants with new-onset epilepsy, of whom 41 (41%) were girls and 59 (59%) were boys. Median age of seizure onset was 128 days (IQR 46-192). For 43 (43% [binomial distribution 95% CI 33-53]) of 100 infants, we identified genetic diagnoses, with a median time from seizure onset to rapid genome sequencing result of 37 days (IQR 25-59). Genetic diagnosis was associated with neonatal seizure onset versus infantile seizure onset (14 [74%] of 19 vs 29 [36%] of 81; p=0·0027), referral setting (12 [71%] of 17 for intensive care, 19 [44%] of 43 non-intensive care inpatient, and 12 [28%] of 40 outpatient; p=0·0178), and epilepsy syndrome (13 [87%] of 15 for self-limited epilepsies, 18 [35%] of 51 for developmental and epileptic encephalopathies, 12 [35%] of 34 for other syndromes; p=0·001). Rapid genome sequencing revealed genetic heterogeneity, with 34 unique genes or genomic regions implicated. Genetic diagnoses had immediate clinical utility, informing treatment (24 [56%] of 43), additional evaluation (28 [65%]), prognosis (37 [86%]), and recurrence risk counselling (all cases). INTERPRETATION: Our findings support the feasibility of implementation of rapid genome sequencing in the clinical care of infants with new-onset epilepsy. Longitudinal follow-up is needed to further assess the role of rapid genetic diagnosis in improving clinical, quality-of-life, and economic outcomes. FUNDING: American Academy of Pediatrics, Boston Children's Hospital Children's Rare Disease Cohorts Initiative, Canadian Institutes of Health Research, Epilepsy Canada, Feiga Bresver Academic Foundation, Great Ormond Street Hospital Charity, Medical Research Council, Murdoch Children's Research Institute, National Institute of Child Health and Human Development, National Institute for Health and Care Research Great Ormond Street Hospital Biomedical Research Centre, One8 Foundation, Ontario Brain Institute, Robinson Family Initiative for Transformational Research, The Royal Children's Hospital Foundation, University of Toronto McLaughlin Centre

Understanding entrepreneurship: Challenging dominant perspectives and theorizing entrepreneurship through new postpositivist epistemologies

Entrepreneurship is characterized by complex, dynamic and emergent processes, and the interplay between actors, processes, and contexts. Postpositivistic approaches offer the opportunity to examine subtleties of the phenomenon of entrepreneurship by placing emphasis on a range of its dimensions and the interplays between dimensions. Despite a growing body of postpositivistic research in response to such calls, the legitimacy of these approaches is still subject to debate on the grounds of rigor and relevance. This special issue challenges these prevailing but often hidden assumptions governing the conduct and publication of scholarly inquiry in the field of entrepreneurship and offers alternative perspectives for future research. © 2014 International Council for Small Business

Phosphorylation of CaMKII at Thr253 occurs in vivo and enhances binding to isolated postsynaptic densities

Autophosphorylation of Ca²⁺-calmodulin stimulated protein kinase II (CaMKII) at two sites (Thr286 and Thr305/306) is known to regulate the subcellular location and activity of this enzyme in vivo. CaMKII is also known to be autophosphorylated at Thr253 in vitro but the functional effect of phosphorylation at this site and whether it occurs in vivo, is not known. Using antibodies that specifically recognize CaMKII phosphorylated at Thr253 together with FLAG-tagged wild type and phospho- and dephospho-mimic mutants of α-CaMKII, we have shown that Thr253 phosphorylation has no effect on either the Ca²⁺-calmodulin dependent or autonomous kinase activity of recombinant α-CaMKII in vitro. However, the Thr253Asp phosphomimic mutation increased α-CaMKII binding to subcellular fractions enriched in post-synaptic densities (PSDs). The increase in binding was similar in extent, and additive, to that produced by phosphorylation of Thr286. Thr253 phosphorylation was dynamically regulated in intact hippocampal slices. KCl induced depolarisation increased Thr253 phosphorylation and the phospho-Thr253-CaMKII was specifically recovered in the subcellular fraction enriched in PSDs. These results identify Thr253 as an additional site at which CaMKII is phosphorylated in vivo and suggest that this dynamic phosphorylation may regulate CaMKII function by altering its distribution within the cell

Mass and galaxy distributions of four massive galaxy clusters from Dark Energy Survey Science Verification data

We measure the weak lensing masses and galaxy distributions of four massive galaxy clusters observed during the Science Verification phase of the Dark Energy Survey (DES). This pathfinder study is meant to (1) validate the Dark Energy Camera (DECam) imager for the task of measuring weak lensing shapes, and (2) utilize DECam’s large field of view to map out the clusters and their environments over 90 arcmin. We conduct a series of rigorous tests on astrometry, photometry, image quality, point spread function (PSF) modelling, and shear measurement accuracy to single out flaws in the data and also to identify the optimal data processing steps and parameters. We find Science Verification data from DECam to be suitable for the lensing analysis described in this paper. The PSF is generally well behaved, but the modelling is rendered difficult by a flux-dependent PSF width and ellipticity. We employ photometric redshifts to distinguish between foreground and background galaxies, and a red-sequence cluster finder to provide cluster richness estimates and cluster–galaxy distributions. By fitting Navarro–Frenk–White profiles to the clusters in this study, we determine weak lensing masses that are in agreement with previous work. For Abell 3261, we provide the first estimates of redshift, weak lensing mass, and richness. In addition, the cluster–galaxy distributions indicate the presence of filamentary structures attached to 1E 0657−56 and RXC J2248.7−4431, stretching out as far as 1◦(approximately 20 Mpc), showcasing the potential of DECam and DES for detailed studies of degree-scale features on the sky