Quantitative Analysis of Focused A-To-I RNA Editing Sites by Ultra-High-Throughput Sequencing in Psychiatric Disorders

A-to-I RNA editing is a post-transcriptional modification of single nucleotides in RNA by adenosine deamination, which thereby diversifies the gene products encoded in the genome. Thousands of potential RNA editing sites have been identified by recent studies (e.g. see Li et al, Science 2009); however, only a handful of these sites have been independently confirmed. Here, we systematically and quantitatively examined 109 putative coding region A-to-I RNA editing sites in three sets of normal human brain samples by ultra-high-throughput sequencing (uHTS). Forty of 109 putative sites, including 25 previously confirmed sites, were validated as truly edited in our brain samples, suggesting an overestimation of A-to-I RNA editing in these putative sites by Li et al (2009). To evaluate RNA editing in human disease, we analyzed 29 of the confirmed sites in subjects with major depressive disorder and schizophrenia using uHTS. In striking contrast to many prior studies, we did not find significant alterations in the frequency of RNA editing at any of the editing sites in samples from these patients, including within the 5HT2C serotonin receptor (HTR2C). Our results indicate that uHTS is a fast, quantitative and high-throughput method to assess RNA editing in human physiology and disease and that many prior studies of RNA editing may overestimate both the extent and disease-related variability of RNA editing at the sites we examined in the human brain

An infrared optical pacing system for screening cardiac electrophysiology in human cardiomyocytes

Human cardiac myocytes derived from pluripotent stem cells (hCM) have invigorated interest in genetic disease mechanisms and cardiac safety testing; however, the technology to fully assess electrophysiological function in an assay that is amenable to high throughput screening has lagged. We describe a fully contactless system using optical pacing with an infrared (IR) laser and multi-site high fidelity fluorescence imaging to assess multiple electrophysiological parameters from hCM monolayers in a standard 96-well plate. Simultaneous multi-site action potentials (FluoVolt) or Ca2+ transients (Fluo4-AM) were measured, from which high resolution maps of conduction velocity and action potential duration (APD) were obtained in a single well. Energy thresholds for optical pacing were determined for cell plating density, laser spot size, pulse width, and wavelength and found to be within ranges reported previously for reliable pacing. Action potentials measured using FluoVolt and a microelectrode exhibited the same morphology and rate of depolarization. Importantly, we show that this can be achieved accurately with minimal damage to hCM due to optical pacing or fluorescence excitation. Finally, using this assay we demonstrate that hCM exhibit reproducible changes in repolarization and impulse conduction velocity for Flecainide and Quinidine, two well described reference compounds. In conclusion, we demonstrate a high fidelity electrophysiological screening assay that incorporates optical pacing with IR light to control beating rate of hCM monolayers

Effect of optical pacing on action potential upstroke.

<p>Top shows action potential upstrokes normalized to a 100 mV action potential that were recorded within 0.5 mm of the stimulation site, before pacing began (baseline, 0 min) and then after continuous pacing at 5 min, 10 min, 15min, 20 min, and 25 min. Bottom shows maximum dVm/dt during the upstroke for sites within 0.5 mm of the stimulation site (n = 4). No significant differences were observed between baseline (0 min) and all other time points.</p

System validation using Flecainide and Quinidine.

<p>Activation time (top) and APD (bottom) contour maps measured at baseline (left) and after 0.3 μM Flecainide (right) in a single well of a 96-well plate. Mean local conduction velocity and APD for each map are shown below. Site of optical pacing is shown by red spot in activation contours. Flecainide decreased conduction velocity and increased APD. To the right are summary data for mean local conduction velocity (top) and APD (bottom) before (CNTL) and after Flecainide (n = 7, n = 6) and Quinidine (1.0 μM, n = 7).</p

Demonstration of optical pacing and activation recording.

<p>Optical pacing in an hCM monolayer from a single well of a 96-well plate. Top shows an action potential recording (Vm) and a Ca2+ transient recording from a single pixel in separate wells at room temperature (RT). Middle shows action potentials recorded at 34°C with faster pacing rates. Bottom shows an activation map during optical pacing at 0.5 Hz indicating the local time of maximum Vm derivative (dVm/dt) during the action potential upstroke. Vectors superimposed on contours represents local impulse conduction velocity. The laser spot (red, 400 μm) was imaged while temporarily using a visible wavelength to shows the location of pacing.</p

Measurement of threshold energies for optical pacing.

<p>(A) Assessment of threshold by a search pattern through a fixed number of monolayers, N = 30 total. To compare threshold under different conditions, 50% capture probability was calculated (see text for details). (B) Cell density at three different values (3.1 x 10², 1.0 x 10³ and 2.0 x 10³ cells/mm²), N = 30 for each point. (C) Pulse widths were varied between 5 ms and 40 ms, N = 18 for each point. (D) Spot size was varied between 4 x 10⁻⁴ and 2.9 x 10⁻³ cm², N = 30 for each point. Thresholds required to achieve 50% pacing probability are plotted against radiant exposure per pulse (B, D) and irradiance (C). Error bars reflect the standard error (A) and standard deviation (B-D). In panels C and D, * indicates a statistically significance (p < 0.001) decrease compared to the smallest pulse width and spot size.</p

Comparison between electrical and optical measurements of action potentials.

<p>Comparison of action potential measured using FluoVolt (cyan) and a sharp microelectrode (orange) simultaneously (top) and at a much higher resolution showing the action potential upstroke (middle). All action potential recordings are normalized to a 100 mV amplitude. In both plots, action potentials recorded with FluoVolt and a sharp microelectrode are highly correlated. The action potential upstroke derivative (dVm/dt) for these examples (bottom left) and over all recordings were identical.</p

Quantitative Analysis of Focused A-To-I RNA Editing Sites by Ultra-High-Throughput Sequencing in Psychiatric Disorders

<div><p>A-to-I RNA editing is a post-transcriptional modification of single nucleotides in RNA by adenosine deamination, which thereby diversifies the gene products encoded in the genome. Thousands of potential RNA editing sites have been identified by recent studies (e.g. see Li et al, <em>Science</em> 2009); however, only a handful of these sites have been independently confirmed. Here, we systematically and quantitatively examined 109 putative coding region A-to-I RNA editing sites in three sets of normal human brain samples by ultra-high-throughput sequencing (uHTS). Forty of 109 putative sites, including 25 previously confirmed sites, were validated as truly edited in our brain samples, suggesting an overestimation of A-to-I RNA editing in these putative sites by Li et al (2009). To evaluate RNA editing in human disease, we analyzed 29 of the confirmed sites in subjects with major depressive disorder and schizophrenia using uHTS. In striking contrast to many prior studies, we did not find significant alterations in the frequency of RNA editing at any of the editing sites in samples from these patients, including within the 5HT_2C serotonin receptor (<em>HTR2C</em>). Our results indicate that uHTS is a fast, quantitative and high-throughput method to assess RNA editing in human physiology and disease and that many prior studies of RNA editing may overestimate both the extent and disease-related variability of RNA editing at the sites we examined in the human brain.</p> </div

A-to-I RNA editing in brain is not altered in various psychiatric disorders.

<p><b>A</b>. Shows A-to-I RNA editing frequency of 29 sites from category I in psychiatric patients and normal controls, excluding 8 samples with pH <6.1. RNA editing frequency is presented as mean, expressed as a percentage of the total population of transcripts, ± SEM. The data were analyzed by t-test with Benjamini–Hochberg correction for multiple comparisons using a P value of 0.05 as the criterion for statistical significance. The editing frequency in patients did not differ significantly from controls for any site tested. <b>B</b>. Shows the expression patterns of 24 isoforms of the 5HT_2C receptor produced by RNA editing in psychiatric patients and normal controls, excluding 8 samples with pH<6.1. The RNA editing frequency is presented as mean, expressed as a percentage of the total population of transcripts, ± SEM. The data were analyzed by s t-test with Benjamini–Hochberg correction for multiple comparisons using a P value of 0.05 as criterion of statistical significance. Editing frequency in patients did not differ significantly from controls for any site tested. <b>C</b>. Shows the frequency of A-to-I RNA editing of 29 sites from category I was examined in 8 samples with pH<6.1 and 8 matched samples with pH ≥6.1. The RNA editing frequency is presented as mean, expressed as a percentage of the total population of transcripts, ± SEM. The data were analyzed by t-test with Benjamini–Hochberg correction. Significant differences between the normal pH group and low pH group are shown by asterisks (*p<0.05; **p<0.01), and were seen in 6 of 29 sites examined.</p