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

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

Summary of Illumina sequencing data.

a<p>Coverage is the number of reads covered in each site, presented with Mean ± SEM.</p>b<p>A-to-G error rate was measured by quantifying the frequency of A-to-G misreads in unedited sites, presented with Mean ± SEM.</p

A-to-I RNA editing is different between the cortex and cerebellum, but not different between two regions of cortex, in human brain.

<p><b>A</b>. Shows the distribution of A-to-I RNA editing frequency of 36 sites from category I, including 11 of the ‘new’ sites identified by Li et al (ref 24) and 25 previously ‘known’ sites, in the cortex and cerebellum from two sets of normal human brain samples. The RNA editing frequency is presented as a percentage of the total population of transcripts. A two-fold increase of RNA editing frequency at 6 sites (CCNI (R/G), CCNI (K/R), 5HT2C (siteB), 5HT2C (siteC), 5HT2C (siteE) and TRO) in cortex and one site (FLNB) in cerebellum are shown in the inset. <b>B</b>. Shows the distribution of A-to-I RNA editing frequency of 36 sites from category I, in the anterior temporal cortex (ATC) and the right frontal cortex (RFC) from the third set of normal human samples (n = 5). The RNA editing frequency is presented as a percentage of the total population of transcripts. No significant difference was found between these two regions of cortex.</p

Ultra-high-throughput sequencing of potential A-to-I RNA editing sites.

<p><b>A</b>. Shows a schematic diagram of processing and measuring RNA editing using ultra High Throughput Sequencing technology. <b>B</b>. Shows the frequency distribution of sequencing coverage for each editing site. The reads for each editing site from three sets of normal human brain samples were grouped in intervals of 5,000 reads. Using the D’Agostino-Pearson normality test, the data do not differ significantly from a Gaussian distribution. <b>C</b>. Shows the A-to-I RNA editing frequency of 36 sites from category I, including 11 ‘new’ sites and 25 known sites in three sets of normal human samples. RNA editing frequency is presented as mean, expressed as a percentage of the total population of transcripts, ± SEM.</p

A-to-I RNA editing sites with the frequency of editing above 1% in three sets of normal human samples.

a<p>Genomic position is the position in human genomic database from UCSC (<a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>, hg18 version, March 2006 assembly).</p>b<p>Frequency of RNA editing is presented as the percentage of the total population of transcripts.</p>c<p>Reads is the number of transcripts sequenced.</p>d<p>11 new RNA editing sites identified by Li et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043227#pone.0043227-Li1" target="_blank">[24]</a>.</p