The severe epilepsy syndromes of infancy: A population-based study.

OBJECTIVE: To study the epilepsy syndromes among the severe epilepsies of infancy and assess their incidence, etiologies, and outcomes. METHODS: A population-based cohort study was undertaken of severe epilepsies with onset before age 18 months in Victoria, Australia. Two epileptologists reviewed clinical features, seizure videos, and electroencephalograms to diagnose International League Against Epilepsy epilepsy syndromes. Incidence, etiologies, and outcomes at age 2 years were determined. RESULTS: Seventy-three of 114 (64%) infants fulfilled diagnostic criteria for epilepsy syndromes at presentation, and 16 (14%) had "variants" of epilepsy syndromes in which there was one missing or different feature, or where all classical features had not yet emerged. West syndrome (WS) and "WS-like" epilepsy (infantile spasms without hypsarrhythmia or modified hypsarrhythmia) were the most common syndromes, with a combined incidence of 32.7/100 000 live births/year. The incidence of epilepsy of infancy with migrating focal seizures (EIMFS) was 4.5/100 000 and of early infantile epileptic encephalopathy (EIEE) was 3.6/100 000. Structural etiologies were common in "WS-like" epilepsy (100%), unifocal epilepsy (83%), and WS (39%), whereas single gene disorders predominated in EIMFS, EIEE, and Dravet syndrome. Eighteen (16%) infants died before age 2 years. Development was delayed or borderline in 85 of 96 (89%) survivors, being severe-profound in 40 of 96 (42%). All infants with EIEE or EIMFS had severe-profound delay or were deceased, but only 19 of 64 (30%) infants with WS, "WS-like," or "unifocal epilepsy" had severe-profound delay, and only two of 64 (3%) were deceased. SIGNIFICANCE: Three quarters of severe epilepsies of infancy could be assigned an epilepsy syndrome or "variant syndrome" at presentation. In this era of genomic testing and advanced brain imaging, diagnosing epilepsy syndromes at presentation remains clinically useful for guiding etiologic investigation, initial treatment, and prognostication.

Feasibility of Ultra-Rapid Exome Sequencing in Critically Ill Infants and Children With Suspected Monogenic Conditions in the Australian Public Health Care System.

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.

Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line.

Mutations in PARK2 (parkin) can result in Parkinson's disease (PD). Parkin shares a bidirectional promoter with parkin coregulated gene (PACRG) and the transcriptional start sites are separated by only ~200 bp. Bidirectionally regulated genes have been shown to function in common biological pathways. Mice lacking parkin have largely failed to recapitulate the dopaminergic neuronal loss and movement impairments seen in individuals with parkin-mediated PD. We aimed to investigate the function of PACRG and test the hypothesis that parkin and PACRG function in a common pathway by generating and characterizing two novel knockout mouse lines harbouring loss of both parkin and Pacrg or Pacrg alone. Successful modification of the targeted allele was confirmed at the genomic, transcriptional and steady state protein levels for both genes. At 18-20 months of age, there were no significant differences in the behaviour of parental and mutant lines when assessed by openfield, rotarod and balance beam. Subsequent neuropathological examination suggested there was no gross abnormality of the dopaminergic system in the substantia nigra and no significant difference in the number of dopaminergic neurons in either knockout model compared to wildtype mice.

A population-based cost-effectiveness study of early genetic testing in severe epilepsies of infancy.

OBJECTIVE: The severe epilepsies of infancy (SEI) are a devastating group of disorders that pose a major care and economic burden on society; early diagnosis is critical for optimal management. This study sought to determine the incidence and etiologies of SEI, and model the yield and cost-effectiveness of early genetic testing. METHODS: A population-based study was undertaken of the incidence, etiologies, and cost-effectiveness of a whole exome sequencing-based gene panel (targeted WES) in infants with SEI born during 2011-2013, identified through electroencephalography (EEG) and neonatal databases. SEI was defined as seizure onset before age 18 months, frequent seizures, epileptiform EEG, and failure of ≥2 antiepileptic drugs. Medical records, investigations, MRIs, and EEGs were analyzed, and genetic testing was performed if no etiology was identified. Economic modeling was performed to determine yield and cost-effectiveness of investigation of infants with unknown etiology at epilepsy onset, incorporating targeted WES at different stages of the diagnostic pathway. RESULTS: Of 114 infants with SEI (incidence = 54/100 000 live births/y), the etiology was determined in 76 (67%): acquired brain injuries (n = 14), focal cortical dysplasias (n = 14), other brain malformations (n = 17), channelopathies (n = 11), chromosomal (n = 9), metabolic (n = 6), and other genetic (n = 5) disorders. Modeling showed that incorporating targeted WES increased diagnostic yield compared to investigation without targeted WES (48/86 vs 39/86). Early targeted WES had lower total cost ($677 081 U.S. dollars [USD] vs $738 136 USD) than late targeted WES. A pathway with early targeted WES and limited metabolic testing yielded 7 additional diagnoses compared to investigation without targeted WES (46/86 vs 39/86), with lower total cost ($455 597 USD vs $661 103 USD), lower cost per diagnosis ($9904 USD vs $16 951 USD), and a dominant cost-effectiveness ratio. SIGNIFICANCE: Severe epilepsies occur in 1 in 2000 infants, with the etiology identified in two-thirds, most commonly malformative. Early use of targeted WES yields more diagnoses at lower cost. Early genetic diagnosis will enable timely administration of precision medicines, once developed, with the potential to improve long-term outcome.

Meeting the challenges of implementing rapid genomic testing in acute pediatric care.

PURPOSE: The purpose of the study was to implement and prospectively evaluate the outcomes of a rapid genomic diagnosis program at two pediatric tertiary centers. METHODS: Rapid singleton whole-exome sequencing (rWES) was performed in acutely unwell pediatric patients with suspected monogenic disorders. Laboratory and clinical barriers to implementation were addressed through continuous multidisciplinary review of process parameters. Diagnostic and clinical utility and cost-effectiveness of rWES were assessed. RESULTS: Of 40 enrolled patients, 21 (52.5%) received a diagnosis, with median time to report of 16 days (range 9-109 days). A result was provided during the first hospital admission in 28 of 36 inpatients (78%). Clinical management changed in 12 of the 21 diagnosed patients (57%), including the provision of lifesaving treatment, avoidance of invasive biopsies, and palliative care guidance. The cost per diagnosis was AU$13,388 (US$10,453). Additional cost savings from avoidance of planned tests and procedures and reduced length of stay are estimated to be around AU$543,178 (US$424,101). The clear relative advantage of rWES, joint clinical and laboratory leadership, and the creation of a multidisciplinary "rapid team" were key to successful implementation. CONCLUSION: Rapid genomic testing in acute pediatrics is not only feasible but also cost-effective, and has high diagnostic and clinical utility. It requires a whole-of-system approach for successful implementation.

Familial cortical dysplasia caused by mutation in the mammalian target of rapamycin regulator NPRL3.

We describe first cousin sibling pairs with focal epilepsy, one of each pair having focal cortical dysplasia (FCD) IIa. Linkage analysis and whole-exome sequencing identified a heterozygous germline frameshift mutation in the gene encoding nitrogen permease regulator-like 3 (NPRL3). NPRL3 is a component of GAP Activity Towards Rags 1, a negative regulator of the mammalian target of rapamycin complex 1 signaling pathway. Immunostaining of resected brain tissue demonstrated mammalian target of rapamycin activation. Screening of 52 unrelated individuals with FCD identified 2 additional patients with FCDIIa and germline NPRL3 mutations. Similar to DEPDC5, NPRL3 mutations may be considered as causal variants in patients with FCD or magnetic resonance imaging-negative focal epilepsy.

Familial cortical dysplasia type IIA caused by a germline mutation in DEPDC5.

Whole-exome sequencing of two brothers with drug-resistant, early-onset, focal epilepsy secondary to extensive type IIA focal cortical dysplasia identified a paternally inherited, nonsense variant of DEPDC5 (c.C1663T, p.Arg555*). This variant has previously been reported to cause familial focal epilepsy with variable foci in patients with normal brain imaging. Immunostaining of resected brain tissue from both brothers demonstrated mammalian target of rapamycin (mTOR) activation. This report shows the histopathological features of cortical dysplasia associated with a DEPDC5 mutation, confirms mTOR dysregulation in the malformed tissue and expands the spectrum of neurological manifestations of DEPDC5 mutations to include severe phenotypes with large areas of cortical malformation.

Mutations in RAB39B cause X-linked intellectual disability and early-onset Parkinson disease with α-synuclein pathology.

Advances in understanding the etiology of Parkinson disease have been driven by the identification of causative mutations in families. Genetic analysis of an Australian family with three males displaying clinical features of early-onset parkinsonism and intellectual disability identified a ∼45 kb deletion resulting in the complete loss of RAB39B. We subsequently identified a missense mutation (c.503C>A [p.Thr168Lys]) in RAB39B in an unrelated Wisconsin kindred affected by a similar clinical phenotype. In silico and in vitro studies demonstrated that the mutation destabilized the protein, consistent with loss of function. In vitro small-hairpin-RNA-mediated knockdown of Rab39b resulted in a reduction in the density of α-synuclein immunoreactive puncta in dendritic processes of cultured neurons. In addition, in multiple cell models, we demonstrated that knockdown of Rab39b was associated with reduced steady-state levels of α-synuclein. Post mortem studies demonstrated that loss of RAB39B resulted in pathologically confirmed Parkinson disease. There was extensive dopaminergic neuron loss in the substantia nigra and widespread classic Lewy body pathology. Additional pathological features included cortical Lewy bodies, brain iron accumulation, tau immunoreactivity, and axonal spheroids. Overall, we have shown that loss-of-function mutations in RAB39B cause intellectual disability and pathologically confirmed early-onset Parkinson disease. The loss of RAB39B results in dysregulation of α-synuclein homeostasis and a spectrum of neuropathological features that implicate RAB39B in the pathogenesis of Parkinson disease and potentially other neurodegenerative disorders.