Genome-wide DNA methylation analysis

in Slovenian male suicide victims

Katarina 1
Kouter , Tomaž 2
Zupanc , Alja Videtič Paska 1
1Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
2Institute of Forensic Medicine, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

Contact: katarina.kouter@mf.uni-lj.si

Take-home message:
 Despite a decrease in number of suicides for the past decade Slovenia is sadly still ranked as one of the leading European countries regarding suicide rate.
 Suicidal behaviour is a result of interplay between hereditary and environmental factors, tied together by epigenetics. Numerous studies so far have linked epigenetic
mechanisms to suicidal behaviour.
 With the help of NGS (reduced representation bisulfite sequencing; RRBS) we were able to perform the first global analysis of methylation in a highly specified group of
Slovenian male suicide victims who died by hanging.
 Our study included two brain regions; hipocampus (12 subjects) and Brodmann area 9 (18 subjects).
 We obtained methylation information of CpG islands, promoter and other regulatory regions. The global differential methylation pattern showed hypomethylation in
suicide victims and differentially methylated CpGs in genes involved in serotonergic signalling and transcription factors, as well as the differences in methylation of the
genes associated with schizophrenia and Alzheimer's disease.
 Based on the results we will continue with targeted methylation analysis on a larger sample set and include blood as a peripheral tissue, which could serve as a treatment
biomarker in the future.

Suicide and epigenetics DNA methylation analysis results

Suicide is a complex public health problem of global proportions
and is a result of interplay between hereditary and environmental
factors, tied together by epigenetics. In recent years, focus shifted cytosine

towards research in the field of epigenetics, including DNA

methylation as the most common epigenetic modification in
mammals, which can result in altered mRNA expression.
DNA methylation is a process of adding methyl groups to the 5C
position cytosine, which changes DNA properties and can result in methylcytosine

change of DNA activity. There are two main mechanisms: by prevention of

transcriptional protein binding or by binding of methyl-CpG-binding domaine
proteins. Advances in next generation sequencing now enable us to sequence
large portions of the genome. One of the first studies that investigated DNA
methylation in suicide was carried out on Slovenian subjects by Keller et al. Our
recent studies on suicide related genes showed a significantly lower level of
methylation of BDNF promoter in blood of suicide victims and a decrease in
methylation levels of membrane bound COMT and soluble COMT in
hippocampus of suicide victims

Slovenia: Suicide mortality rate per municipality

2012 – 2016

 Male to female ratio approximately 4:1.

 Most common method is hanging.
 More prevalent in Easter region.
 Average age men 51 years, women 56 years.
Source: NIJZ

Aim and hypothesis

Despite a decrease in number of suicides for the past decade, Slovenia is still
ranked as one of the leading European countries regarding suicide rate.
The aim of our study was to investigate DNA methylation on a global scale in a
highly homozygous male suicide victims who died by hanging and control group
to identify differentially methylated regions. We expected to find differences in
methylation levels and patterns between the two studied groups.

Sample preparation and data analysis S6_HIPO

S8_HIPO
S8_BA9
S3_BA9
C4_BA9
S5_BA9
S7_HIPO S2_BA9
S9_HIPO S9_BA9
S4_HIPO S1_BA9
S7_BA9
S5_HIPO S4_BA9

Since methylation is tissue specific our experiment included two brain regions; C4_HIPO
C3_HIPO
C5_BA9
S6_BA9
C2_BA9
C1_HIPO C9_BA9

hippocampus and Brodmann area 9 (BA9). Altogether 18 subject were included C8_HIPO
C6_HIPO
C7_BA9
C8_BA9
C3_BA9
C1_BA9

in our study. Using RRBS (reduced representation bisulfite sequencing) method C2_HIPO C6_BA9

we sequenced BA9 DNA of 9 suicide victims and 9 controls; and hippocampal

DNA of 6 suicide victims and 6 controls. Briefly, samples went through
proccesses of DNA isolation, MspI cleveage, bsulfite conversion, sze selection hippocampus Brodmann area 9
and next generation sequencing. CD36 47.8% LINC00552 33.7%
Most differentially
CDH12 47.1% CNOT1 30.1%
Data analysis: hypermethylated CpGs
RBFOX3 42.7% LSP153 29.9%
 FastQC Most differentially AASDH -72.2% MMEL1 -31.7%
 TrimGalore hypomethylated CpGs GRIN2B -54.4% COL4A2 -30.6%
 Bismark LRBA -49.6% COLEC11 -29.9%
 R packages methylKit and annotatr

References:
This work was supported by the Slovenian
SI-STAT Data Portal, NIJZ, Statistični urad Republike Slovenije.
Research Agency, program grant no. P1-0104,
S. Roškar, M. Zorko, A. Podlesek, Suicide in Slovenia Between 1997 and 2010. Crisis 36, 126-134 (2015).
Slovenian neuroscience assocoiation SiNAPSA
S. Keller et al., Increased BDNF promoter methylation in the Wernicke area of suicide subjects. Archives of general psychiatry 67, 258-267 (2010).
grant and Young researcher grant.
A. Meissner et al., Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 18, 5868-77 (2005).