T75. SUICIDE AND EPIGENETICS: FROM GENOME-WIDE METHYLATION TO TARGETED MIRNA ANALYSIS

Suicide is a multifactorial, polygenic state that affects millions worldwide. It is a result of interplay between hereditary and environmental factors, tied together by epigenetics. Epigenetics links external/environmental and genetic factors, and describes changes in gene expression that are not the result of changes in the DNA sequence. The three most common epigenetic mechanisms are noncoding RNA, DNA methylation and modifications of histone proteins. The study was performed on Slovenian male suicides that died by hanging. First, genome-wide differential DNA methylation was determined by reduced representation bisulfite sequencing (RRBS) in hippocampus and Brodmann area 9, followed by targeted sequencing of eight neuropsychiatric candidate genes (NR3C1, SLC6A4, HTR1A, TPH2, SKA2, MAOA, GABRA1, and NRIP3) in brain regions (hippocampus, insula, amygdala, Brodmann area 46) and blood. In the second part of the study we investigated miRNAs expression in the Brodmann area 10. To select miRNAs we created an in silico analysis algorithm that would help select the best suitable miRNAs targeting the most interesting genes associated with suicidality. We used databases/web algorithms DIANA microT, miRDB, miRmap, miRWalk, and TargetScan and candidate genes SLC6A4, HTR1A, BDNF, NR3C1, ZNF714, and NRIP3. Based on a prediction algorithm, we have chosen miRNAs that are targeting regulation of the genes listed, and are at the same time being expressed in the brain. By genome-wide approach several differences in methylation level between suicides and controls in both brain regions (> 25% difference in methylation and q-value < 0.01), with gene ontology pointing towards cell structural integrity and nervous system regulation. With gene expression analysis two genes, ZNF714 and NRIP3, were determined also as differentially expressed. By targeted amplicon analysis changes were observed in NR3C1 (insula), HTR1A (insula, blood), SKA2 (insula, blood), MAOA (blood), GABRA1 (insula, blood) and NRIP3 (hippocampus, amygdala). Comparing methylation pattern between blood and brain, similarity was observed between blood and insula for HTR1A. Differential gene expression was determined for NR3C1, SLC6A4 and HTR1A in hippocampus. In miRNA analysis the highest ranking scores were obtained for hsa-miR-4516, hsa-miR-3135b, hsa-miR-124-3p, hsa-miR-129-5p, hsa-miR-27b-3p, hsa-miR-381-3p, hsa-miR-4286. For hsa-miR-4516 and hsa-miR-381-3p a trend for statistical significance was shown. Furthermore, expression of the target genes of these miRNAs pointed out lower expression of NR3C1 in suicides compared to controls, which is in accordance with the available literature results. Results provide an insight into the altered state of DNA methylation and miRNA in suicide. Identified new candidate genes could provide a starting point for further studies on clinical samples with highly expressed suicide risk. Additionally, we have shown that in silico approach could be of further use when planning targeted approach studies, and could help identify set of genes with strong association with suicide.