Project description:The pathophysiology of reversible cerebral vasoconstriction syndrome (RCVS) is elusive. We speculated that circulating miRNAs might participate in the pathogenesis of RCVS and could assist clinical diagnosis. We prospectively recruited 75 RCVS patients (including 20 in discovery cohort, 23 in validation cohort-1, and 32 in the validation cohort-2) together with 76 age- and sex-matched controls. Five miRNAs including miR-130a-3p, miR-130b-3p, let-7a-5p, let-7b-5p andlet-7f-5p were significantly upregulated in patients with RCVS during ictal stage in comparison with that in their remission stage or controls. The combined miRNA panel well differentiated patients from controls (area under curve of Receiver Operating Characteristic curve was 0.906, 0.890 and 0.867 in three different cohorts respectively.) The expression of miR-130a-3p was associated with disruption of blood-brain barrier. Target prediction and pathway enrichment analysis suggested that endothelin-1 (EDN1) and transforming growth factor-beta (TGF-β) signaling pathway might link these miRNAs to the pathogenesis of RCVS. In vitro functional validation in human umbilical vein endothelial cells (HUVEC), human blood-brain barrier cell line (hCMEC/D3 cells) and HEK 293 cells confirmed that EDN1 and genes (BMPR2, SMAD5 and TGFBR2) involved in TGF-β signaling pathway were downregulated by these miRNAs. These miRNAs were correlated with plasma endothelin-1 level in the controls but not patients, indicating a deregulated feedback loop in patients during the disease state. To conclude, we identified the miRNA signatures associated with RCVS, which revealed excellent diagnostic performance, clinical relevance, and were functionally relevant to the putative pathomechanisms.
Project description:Sequence-based genetic testing identifies causative variants in ~50% of individuals with developmental and epileptic encephalopathies (DEEs). Aberrant changes in DNA methylation are implicated in various neurodevelopmental disorders but remain unstudied in DEEs. We interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 582 individuals with genetically unsolved DEEs. We identify rare differentially methylated regions (DMRs) and explanatory episignatures to uncover causative and candidate genetic etiologies in 12 individuals. Using long-read sequencing, we identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and four copy number variants. We also identify pathogenic variants associated with episignatures. Finally, we refine the CHD2 episignature using an 850K methylation array and bisulfite sequencing to investigate potential insights into CHD2 pathophysiology. Our study demonstrates the diagnostic yield of genome-wide DNA methylation analysis to identify causal and candidate variants as 2% (12/582) for unsolved DEE cases.
Project description:Effective management and control of parasitic infections on farms depends on their early detection. Traditional serological diagnostic methods for Fasciola hepatica infection in livestock are specific and sensitive, but currently the earliest detection of the parasite only occurs at approximately three weeks post-infection. At this timepoint, parasites have already entered the liver and caused the tissue damage and immunopathology that results in reduced body weight and loss in productivity. Here, we investigated whether the differential abundance of micro(mi)miRNAs in sera of F. hepatica-infected sheep has potential as a tool for the early diagnosis of infection. Using miRNA sequencing analysis, we discovered specific profiles of sheep miRNAs at both the pre-hepatic and hepatic infection phases in comparison to non-infected sheep. In addition, six F. hepatica-derived miRNAs were specifically identified in sera from infected sheep. Thus, a panel of differentially expressed miRNAs comprising four sheep (miR-3231-3p; miR133-5p; 3957-5p; 1197-3p) and two parasite miRNAs (miR-124-3p; miR-Novel-11-5p) were selected as potential biomarkers. The expression of these candidates in sera samples from longitudinal sheep infection studies collected between 7 days and 23 weeks was quantified using RT-qPCR and compared to samples from age-matched non-infected sheep. We identified oar-miR-133-5p and oar-miR-3957-5p as promising biomarkers of fasciolosis, detecting infection as early as 7 days. The differential expression of the other selected miRNAs was not sufficient to diagnose infection; however, our analysis found that the most abundant forms of fhe-miR-124-3p in sera were sequence variants (IsomiRs) of the canonical miRNA, highlighting the critical importance of primer design for accurate diagnostic RT-qPCR. Accordingly, this investigative study suggests that certain miRNAs are biomarkers of F. hepatica infection and validates miRNA-based diagnostics for the detection of fasciolosis in sheep.