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.

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.

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.

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.

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.