Transcriptomes of Dravet syndrome iPSC derived GABAergic cells reveal dysregulated pathways for chromatin remodeling and neurodevelopment.

Dravet syndrome (DS) is an early onset refractory epilepsy typically caused by de novo heterozygous variants in SCN1A encoding the α-subunit of the neuronal sodium channel Nav1.1. The syndrome is characterized by age-related progression of seizures, cognitive decline and movement disorders. We hypothesized that the distinct neurodevelopmental features in DS are caused by the disruption of molecular pathways in Nav1.1 haploinsufficient cells resulting in perturbed neural differentiation and maturation. Here, we established DS-patient and control induced pluripotent stem cell derived neural progenitor cells (iPSC NPC) and GABAergic inter-neuronal (iPSC GABA) cells. The DS-patient iPSC GABA cells showed a shift in sodium current activation and a perturbed response to induced oxidative stress. Transcriptome analysis revealed specific dysregulations of genes for chromatin structure, mitotic progression, neural plasticity and excitability in DS-patient iPSC NPCs and DS-patient iPSC GABA cells versus controls. The transcription factors FOXM1 and E2F1, positive regulators of the disrupted pathways for histone modification and cell cycle regulation, were markedly up-regulated in DS-iPSC GABA lines. Our study highlights transcriptional changes and disrupted pathways of chromatin remodeling in Nav1.1 haploinsufficient GABAergic cells, providing a molecular framework that overlaps with that of neurodevelopmental disorders and other epilepsies.

Measurement of urinary cystatin C with a particle-enhanced turbidimetric immunoassay on Architect ci8200.

BACKGROUND: Cystatin C is a low-molecular-weight protein that is freely filtered by the glomerulus and catabolized after reabsorption by the proximal tubular cells in healthy subjects. Urinary cystatin C is a potential biomarker for tubular damage including acute kidney injury (AKI) in the acute phase when patients are submitted to the intensive care unit. METHODS: The aim of this study was to perform a method validation of urinary analysis of cystatin C by particle-enhanced turbidimetric immunoassay (PETIA) on a high-throughput chemical analyzer. Total assay time was 10 min. The antigen excess, linearity, lower limit of quantification (LoQ), recovery, assay precision, stability, and interference caused by hemoglobin were evaluated. RESULTS: The LoQ was calculated to 0.020 mg/l with a coefficient of variation (CV) ≤ 10%. No hook effect was observed and the assay was linear over the studied interval less than 0.020-0.950 mg/l with a regression of R² = 0.9994. The assay had a recovery between 93-100% and the assay precision had a total CV of less than 3.5%. Cystatin C was stable for 3 days in room temperature and 14 days in +4C. The assay did not show any major interference with hemoglobin at a hemoglobin concentration of 10 g/L. The reference interval for urine cystatin C was less than 0.166 mg/l. CONCLUSION: The urinary cystatin C PETIA showed good precision and performance characteristics including short test turnaround times that are necessary qualifications for a biomarker at a routine laboratory.

Particle enhanced turbidimetric immunoassay for the determination of urine cystatin C on Cobas c501.

OBJECTIVE: Urinary cystatin C has been reported to be a good marker for tubular damage and acute kidney injury. The aim of this study was to develop a high throughput assay for the quantification of urine cystatin C. METHODS: Antigen-excess, imprecision, interference, linearity, recovery, sample stability and reference values were evaluated on Cobas c501. RESULTS: The assay was linear over the dynamic range of the study (R²=0.9994). The total assay imprecision was below 5%. The assay recovery was estimated at 87-100%. No tendency to antigen-excess (up to 35 mg/L), nor interference with haemoglobin (1.25-10 g/L) was observed. Cystatin C was stable for 1 day at ambient temperature (19-23°C) but for 2 days at +4°C. The reference interval for cystatin C in urine was <0.414 mg/L. CONCLUSIONS: The urinary cystatin C assay verified to be a reliable assay with convenient performance characteristics, enabling routine testing on clinical chemistry platforms.