Table 1. Demographic characteristics and psychiatric diagnoses.1 Male/Female Age (years) PMI (hours) pH Childhood Abuse/Neglect Mood Disorder Alcohol/Drug Abuse Disorder 12/18 Anxiety Disorder Suicide 18/0 34 6 9 24 6 5.3 6.4 6 0.4 18/18 14/18 3/18 100% 78% 67% 17% Control 12/0 36 6 12 23 6 5.9 6.5 6 0.2 0/12 3/12 5/12 1/12 0% 25% 42% 8% The values are mean 6 SD. 1The number of subjects in each group represents the total number of subjects used for methylation and expression analysis. Additional subjects used for expression analysis did not differ from the other subjects in any of the listed measures (see Materials and Methods for details; P’s.0.05). doi:10.1371/journal.pone.0002085.t001 different CpG densities in the core promoter and UCE (3 in the mouse and 26–28 in the human [31,33]). Thus, although in both species complete methylation of CpGs in the promoters charac- terizes inactive alleles, the number of CpGs involved is different suggesting a different mode of regulation by DNA methylation. In the present study, we tested the hypothesis that rRNA in the human hippocampus of suicide subjects with a history of childhood abuse or severe neglect and controls who died suddenly of unrelated causes without a history of childhood abuse or severe neglect is differentially methylated and expressed. Within the genome there are over 400 copies of the rRNA gene, encoding a rRNA Methylation in Suicide large pre-rRNA transcriptional unit (45S) whose expression is tightly regulated by methylation [31–34]. We particularly examined the core promoter region and UCE of rRNA because it is involved in the regulation of all pol I transcribed copies of rRNA by methylation [33]. Our strategy was to sample the average methylation pattern of the rRNA promoter at single nucleotide resolution to determine CpG site specificity in the regulation of rRNA gene expression in the brains of suicide subjects and controls. The results implicate the epigenetic modulation of rRNA in the pathophysiology of suicide. Results The subject characteristics are presented in Table 1. There were no significant differences in post-mortem interval (PMI), brain pH, or age between suicide subjects and controls (P’s.0.05). Genotyping of the rRNA promoter Because alterations in rRNA function may occur due to both genetic and epigenetic differences, the rRNA promoter region from each suicide subject and control was sequenced. No sequence variants were seen among subjects (Fig. 1; also see Fig. S1). When the sequence was compared to the published reference sequence for the rRNA promoter region (Genebank accession number: U13369) a few discrepancies were discovered. Notably, two CpG dinucleotides were not found in the sequenced DNA (Fig. 1). One CpG dinucleotide between the CpG dinucleotides in positions 2108 and 2103 was simply absent, and a second just upstream of the CpG dinucleotide in position 23 was found to be a C/T nucleotide substitution. Thus, of the 28 CpG dinucleotides in the published sequence, 26 CpG dinucleotides were present in the samples. As all subjects in our study were of French-Canadian origin, a population with a well established founder effect [35], it is Figure 1. Genotyping of the rRNA promoter. The rRNA promoter sequence was identical for all suicide subjects and controls. The sequence derived from genotyping is shown above the published rRNA sequence, indicating consensus sequences for primers used for sodium bisulfite mapping (underline) and CpG dinucleotides (bold font), with locations marked relative to the transcription start site (arrow). Differences with the published rRNA sequence, U13369, are highlighted in grey, and the base pair length of each sequence is listed on the right side. doi:10.1371/journal.pone.0002085.g001 PLoS ONE | www.plosone.org 2 May 2008 | Volume 3 | Issue 5 | e2085