Katherine B. Howell

Tissue reoxygenation following hypoxia is associated with ischemia‐reperfusion injury (IRI) and may signal the development of ischemic preconditioning, an adaptive state that is protective against subsequent IRI. Here we used microarray RNA analysis of in vivo and in vitro models of IRI to delineate the underlying molecular mechanisms. Microarray analysis of renal tissue after ischemia‐reperfusion revealed a number of highly up‐regulated antioxidant genes including aldehyde dehydrogenases (ALDH1A1 and ALDH1A7), glutathione S‐transferases (GSTM5, GSTA2 and GSTP1), and NAD(P)H quinone oxidoreductase (NQO1). The transcription factor NF‐E2‐related factor‐2 (Nrf2), a master regulator of this antioxidant response, is also elevated in IRI. Further‐more, microarray analysis of renal epithelial cells exposed to hypoxia/reoxygenation identifi Nrf2 to be up‐regulated on reoxygenation. We also reveal a reoxygenation‐specific nuclear accumulation of Nrf2 protein and subsequent activation of a NQO1 promoter reporter construct. Attenuating reactive oxygen species (ROS) in reoxygenation using the antioxidant N‐acetyl cysteine results in inhibition of Nrf‐2 activation. mRNA levels for Nrf2‐dependent genes were detected in human liver biopsy 1 h after transplantation. These results indicate that reoxygenation‐dependent Nrf‐2 activity facilitates ischemic preconditioning through the induction of antioxidant gene expression and that ROS may be critical in signaling this event.—Leonard, M. O., Kieran, N. E., Howell, K., Burne, M. J., Varadarajan, R., Dhakshinamoorthy, S., Porter, A. G., O'Farrelly, C., Rabb, H., Taylor, C. T. Reoxygenation‐specific activation of the antioxidant transcription factor Nrf2 mediates cytoprotective gene expression in ischemia reperfusion injury. FASEB J. 20, E2166–E2176 (2006)

OBJECTIVE: De novo SCN2A mutations have recently been associated with severe infantile-onset epilepsies. Herein, we define the phenotypic spectrum of SCN2A encephalopathy. METHODS: Twelve patients with an SCN2A epileptic encephalopathy underwent electroclinical phenotyping. RESULTS: Patients were aged 0.7 to 22 years; 3 were deceased. Seizures commenced on day 1-4 in 8, week 2-6 in 2, and after 1 year in 2. Characteristic features included clusters of brief focal seizures with multiple hourly (9 patients), multiple daily (2), or multiple weekly (1) seizures, peaking at maximal frequency within 3 months of onset. Multifocal interictal epileptiform discharges were seen in all. Three of 12 patients had infantile spasms. The epileptic syndrome at presentation was epilepsy of infancy with migrating focal seizures (EIMFS) in 7 and Ohtahara syndrome in 2. Nine patients had improved seizure control with sodium channel blockers including supratherapeutic or high therapeutic phenytoin levels in 5. Eight had severe to profound developmental impairment. Other features included movement disorders (10), axial hypotonia (11) with intermittent or persistent appendicular spasticity, early handedness, and severe gastrointestinal symptoms. Mutations arose de novo in 11 patients; paternal DNA was unavailable in one. CONCLUSIONS: Review of our 12 and 34 other reported cases of SCN2A encephalopathy suggests 3 phenotypes: neonatal-infantile-onset groups with severe and intermediate outcomes, and a childhood-onset group. Here, we show that SCN2A is the second most common cause of EIMFS and, importantly, does not always have a poor developmental outcome. Sodium channel blockers, particularly phenytoin, may improve seizure control.

Importance: Widespread adoption of rapid genomic testing in pediatric critical care requires robust clinical and laboratory pathways that provide equitable and consistent service across health care systems. Objective: To prospectively evaluate the performance of a multicenter network for ultra-rapid genomic diagnosis in a public health care system. Design, Setting, and Participants: Descriptive feasibility study of critically ill pediatric patients with suspected monogenic conditions treated at 12 Australian hospitals between March 2018 and February 2019, with data collected to May 2019. A formal implementation strategy emphasizing communication and feedback, standardized processes, coordination, distributed leadership, and collective learning was used to facilitate adoption. Exposures: Ultra-rapid exome sequencing. Main Outcomes and Measures: The primary outcome was time from sample receipt to ultra-rapid exome sequencing report. The secondary outcomes were the molecular diagnostic yield, the change in clinical management after the ultra-rapid exome sequencing report, the time from hospital admission to the laboratory report, and the proportion of laboratory reports returned prior to death or hospital discharge. Results: The study population included 108 patients with a median age of 28 days (range, 0 days to 17 years); 34% were female; and 57% were from neonatal intensive care units, 33% were from pediatric intensive care units, and 9% were from other hospital wards. The mean time from sample receipt to ultra-rapid exome sequencing report was 3.3 days (95% CI, 3.2-3.5 days) and the median time was 3 days (range, 2-7 days). The mean time from hospital admission to ultra-rapid exome sequencing report was 17.5 days (95% CI, 14.6-21.1 days) and 93 reports (86%) were issued prior to death or hospital discharge. A molecular diagnosis was established in 55 patients (51%). Eleven diagnoses (20%) resulted from using the following approaches to augment standard exome sequencing analysis: mitochondrial genome sequencing analysis, exome sequencing-based copy number analysis, use of international databases to identify novel gene-disease associations, and additional phenotyping and RNA analysis. In 42 of 55 patients (76%) with a molecular diagnosis and 6 of 53 patients (11%) without a molecular diagnosis, the ultra-rapid exome sequencing result was considered as having influenced clinical management. Targeted treatments were initiated in 12 patients (11%), treatment was redirected toward palliative care in 14 patients (13%), and surveillance for specific complications was initiated in 19 patients (18%). Conclusions and Relevance: This study suggests feasibility of ultra-rapid genomic testing in critically ill pediatric patients with suspected monogenic conditions in the Australian public health care system. However, further research is needed to understand the clinical value of such testing, and the generalizability of the findings to other health care settings.