rRNA Methylation in Suicide Figure 6. rRNA expression is downregulated in suicide. (A) Suicide subjects (N = 16, black bar) showed significantly less rRNA expression than controls (N = 9, white bar); Data expressed as mean 6 S.E.M. *, P,0.05, measured by unpaired t-test. (B) Among subjects where both methylation and expression of rRNA were analyzed, the correlation between rRNA promoter methylation percentage and expression showed a trend for an inverse relationship between methylation percentage and expression of rRNA in suicide subjects (N = 11, filled circles) and controls (N = 8, open circles). doi:10.1371/journal.pone.0002085.g006 In summary, these data reveal increased promoter-wide methylation of the rRNA promoter as well as decreased rRNA gene expression in suicide subjects. The results of the psychological autopsy suggest a developmental origin, however, at time this is speculation based on samples that differ along a wide range of experiential and potentially genetic dimensions. To date, our data are merely consistent with the hypothesis that early life events can alter the epigenetic status of genes that mediate neural functions, and thus contribute to individual differences in the risk for suicide. Materials and Methods Subjects and Sample Preparation Hippocampal samples obtained from the Quebec Suicide Brain Bank included: 13 suicide subjects and 11 controls matched for post-mortem interval (PMI), gender, and age (Table 1). Cerebel- lar samples were also obtained for 4 of the same suicide subjects and 4 of the same controls as those used for hippocampal analysis. An additional 6 hippocampal samples consisting of 5 suicide subjects and 1 control were also obtained from the Quebec Suicide Brain Bank for RNA expression analysis, to compensate for the removal of 5 subjects (2 suicide and 3 controls) due to low RNA quality. All samples were from male suicide and control subjects of French-Canadian origin that were processed as previously described [50]. Samples were dissected at 4uC and stored in plastic vials at 280uC until analysis. All samples were processed and analyzed blind to demographic and diagnostic variables. Possible confounders that were examined included PMI, brain pH, and age at death of the donor (Table 1). This study was approved by our local Institutional Review Board and signed informed consent was obtained from next of kin. To be included in this study, all subjects had to die suddenly, with no medical or paramedic intervention. Suicide as the cause of death was determined by the Quebec Coroner’s Office. Psychi- atric diagnoses were obtained by means of the SCID I [51] and SCID II [52] interviews adapted for psychological autopsies, which is a validated method to reconstruct psychiatric history by means of extensive proxy-based interviews, as outlined elsewhere [41]. In addition, to be considered in this study, all suicide subjects and none of the controls had to have a positive history of childhood abuse or severe neglect, as determined by structured interviews using the Childhood Experience of Care and Abuse (CECA) [53] questionnaire adapted for psychological autopsies, as described elsewhere [54]. Genotyping of the rRNA promoter region Genomic DNA was extracted (DNeasy, Qiagen) according to the manufacturer’s protocol. Primers for PCR were directed against the rRNA gene promoter (Genebank accession number U13369) using the following sequences: 59-GTG TGT CCC GGT CGT AGG-39; antisense: 59-GTC ACC GTG AGG CCA GAG- 39. Primers were selected on the basis that they covered a 400bp region that included the region selected for sodium bisulfite analysis, including the regions covered by sodium bisulfite primers. The resulting PCR products for each subject were sequenced bidirectionally using the forward and the reverse primer on an ABI 3100 genetic analyzer (Applied Biosystems) and following the manufacturer’s instructions. Genetic variation was assessed throughout the rRNA promoter region used for bisulfite analysis by alignment of genomic DNA with the previously published rRNA gene promoter sequence using freely available software (CLC Workbench, CLC bio). PLoS ONE | www.plosone.org 7 May 2008 | Volume 3 | Issue 5 | e2085