GENOME-WIDE DNA METHYLATION PATTERNS IN SUICIDE VICTIMS: IDENTIFICATION OF NEW CANDIDATE GENES

Suicide is a major global public health problem with significant impact on society. According to the World Health Organization, every year about 800.000 people commit suicide, while at the global level suicide accounts for 50 % of all violent deaths among men and for 71 % among women. Suicide is a complex phenomenon which cannot be attributed to a single causal factor, but to a combination of simultaneous effects of multiple factors which are expressed in the form of psychological, biological and sociological indicators. Analysis of epigenetic mechanisms (methylation of the DNA, modifications of histone proteins and (networks of) miRNA), which link the interaction between genes and the environment, could importantly contribute to better understanding of suicidal behaviour. Recent studies on suicidal behaviour and DNA methylation show differences in DNA methylation pattern, with numerous sites among suicide victims. Using next generation sequencing, genome-wide studies helped identify novel candidate genes while studies of already known candidate genes (such as glucocorticoid receptor and BDNF) gave us better insight into the interplay of genetics and epigenetics. Epigenetic studies importantly contribute to elucidation of new biomarkers for suicidal behaviour. However, present studies are very different in design and often performed on very small samples, and these limitations could be overcome with more careful study preparation

DNA methylation in Slovenian male suicide victims by hanging

Samomorilno vedenje je poligensko, večfaktorsko vedenje, ki prizadane ljudi širom sveta. Na samomorilno vedenje vplivajo številni biološki in zunanji dejavniki, ki jih lahko povežemo z epigenetiko. Mehanizmi in dejavniki samomorilnega vedenja kljub širokemu naboru novih dognanj niso v celoti poznani. Na podlagi molekularno-bioloških študij pa lahko sklepamo, da obstaja tudi pomembna biološka komponenta samomorilnega vedenja. Ena izmed njih je metilacija DNA in spremenjena stopnja le-te. Da bi preverili, ali obstajajo razlike v stopnji metilacije, smo tvorili dve skupini, ki smu ju primerjali med seboj. Prva je zajemala moške, ki so umrli zaradi samomora z obešanjemdruga skupina pa je predstavljala kontrolno skupino (zajemala je moške, ki so umrli zaradi nenadne srčne smrti). Z uporabo tehnologije sekvenciranja naslednje generacije (NGS) smo v prvem delu preiskovali stopnjo metilacije DNA širom celotnega genoma na manjšem naboru preiskovancev (v možganskih regijah hipokampus in Brodmannovo področje (BA) 9). V drugem delu smo na celotni skupini preiskovancev preverili stopnjo metilacije tarčnih zaporedij DNA izbranih kandidatnih genov (možganske regije amigdala, BA46, hipokampus in insula ter kri). Rezultate, pridobljene z NGS smo želeli potrditi z dodatno metodo. Kandidatne gene, pri katerih smo opazili spremenjeno stopnjo metilacije med obema preiskovanima skupinama, smo dodatno preučili z analizo njihovega izražanja s qPCR tako za prvi kot tudi za drugi del doktorske naloge. Pokazali smo številne razlike v stopnji metilacije med žrtvami samomora in kontrolno skupino. V prvem delu smo z analizo metilacije DNA širom genoma pri obeh preiskovanih možganskih regijah našli večje število diferenčno metiliranih citozinov z razliko v odstotku metilacije med obema skupinama preiskovancev večjo od 25 % in q-vrednostjo pod 0,01. V analizi genske ontologije so bili obogateni izrazi, povezani s strukturno integriteto celice in z uravnavanjem živčnega sistema. V analizi izražanja genov smo v BA9 pri žrtvah samomora opazili povišano izražanje dveh genov (ZNF714 in NRIP3). V drugem delu, analizi metilacije tarčnih zaporedij DNA, smo prav tako opazili razlike v stopnji metilacije med preiskovanima skupinama. Razlike smo opazili v vseh sekvenciranih kandidatnih genih, pri čemer pa so bile nekatere razlike v odstotku metilacije nizke (nižje od enega odstotka). Spremenjeno izražanje genov smo opazili v hipokampusu (ZNF714, NR3C1, SLC6A4 in HTR1A) in krvi (NRIP3). Rezultati naših analiz dajejo nov uvid v spremenjeno stanje metilacije pri samomorilnem vedenju in nakazujejo možnost spremenjene plastičnosti možganov. Pridobili smo nove kandidatne gene ter pokazali povezavo s samomorilnim vedenjem pri že obstoječih kandidatnih genih. Rezultati tako podpirajo vlogo biološke komponente ozadja samomorilnega vedenja in prispevajo k osvetljevanju problematike na visoko samomorilno ogroženi slovenski populaciji.Suicidal behaviour is a polygenic and multifactorial disorder affecting people all around the world. It is influenced by multiple factors, which can be tied together by epigenetics. Although comprehensive amount of knowledge on suicidal behaviour has been gathered, complete mechanism and factors leading to suicidal behaviour are yet to be determined. However, based on molecular studies, we can conclude that there is an important biological component to suicidal behaviour, and DNA methylation could play an important role. For the purpose of this work we formed two study groups. First included 25 male suicide victims, who died by hanging. The second group was a control group, including 28 males who died from sudden cardiac arrest. Using advances of the next-generation sequencing technology (NGS), we first analysed genome-wide DNA methylation on a smaller subset. DNA methylation was examined in hippocampus and in a cortical region, Brodmann area (BA) 9. Next, we interrogated DNA methylation of selected candidate genes in additional brain regions (beside hippocampus also amygdala, insula and BA46) and blood using the complete set of subjects. We wanted to confirm the results obtained using NGS with an additional method. Candidate genes for which we observed an altered rate of methylation between the two study groups were further examined by analysing their expression by qPCR for both the first and second part of the doctoral thesis. In our study we determined multiple differences in DNA methylation state between suicide victims and the control group. Genome-wide DNA methylation revealed increased hypomethylation in suicide victims for both brain regions. We found a large number of differentially methylated cytosines with the difference of the percentage of methylation between the two groups greater than 25 % and with the q-value below 0.01. Gene ontology analysis showed enrichment for genes associated with cell structural integrity and nervous system regulation. In BA9 expression of two genes was elevated in suicide victims (ZNF714 and NRIP3). In the second part of our work, we performed DNA methylation analysis for candidate genes, and we detected differences in DNA methylation for all studied brain regions and blood. Although we observed differences within all candidate genes, some differences in DNA methylation were low (less than one percent between studied groups). Gene expression between suicide victims and control group differed statistically in the hippocampus (ZNF714, NR3C1, SLC6A4, and HTR1A) and blood (NRIP3). The results of our analyses provide new insights into the methylation-altered state in suicidal behaviour and suggest the possibility of changes in brain plasticity. We identified new potential candidate genes and strengthen the association with suicidal behaviour in already known candidate genes. The results therefore support the role of the biological component in suicidal behaviour and contribute to its understanding in a highly suicidal Slovenian population

Targeted sequencing approach

Objectives: Epigenetic mechanisms are involved in regulation of many pathologies, including suicidal behaviour. However, the factors through which epigenetics affect suicidal behaviour are not fully understood. Methods: We analysed DNA methylation of eight neuropsychiatric genes (NR3C1, SLC6A4, HTR1A, TPH2, SKA2, MAOA, GABRA1, and NRIP3) in brain regions (hippocampus, insula, amygdala, Brodmann area 46) and blood of 25 male suicide victims and 28 male control subjects, using bisulphite next-generation sequencing. Results: Comparing mean methylation values, notable changes were observed in NR3C1 (insula p-value = 0.05), HTR1A (insula p-value < 0.001, blood p-value = 0.001), SKA2 (insula p-value = 0.03, blood p-value = 0.016), MAOA (blood p-value < 0.001), GABRA1 (insula p-value = 0.05, blood p-value = 0.024) and NRIP3 (hippocampus p-value = 0.001, insula p-value = 0.002, amygdala p-value = 0.014). Comparing methylation pattern between blood and brain, similarity was observed between blood and insula for HTR1A. Gene expression analysis in hippocampus revealed changes in expression of NR3C1 (p-value = 0.049), SLC6A4 (p-value = 0.017) and HTR1A (p-value = 0.053). Conclusions: Results provide an insight into the altered state of DNA methylation in suicidal behaviour. Epigenetic differences could therefore affect suicidal behaviour in both previously known and in novel neuropsychiatric candidate genes

Suicide and changes in expression of neuronal miRNA predicted by an algorithm search through miRNA databases

Suicide is multifactorial and polygenic phenotype, affected by environmental and genetic factors. Among epigenetic mechanisms, miRNAs have been studied, but so far no very concise results exist. To overcome limitations of candidate miRNA and whole genome sequencing approaches, we created an in silico analysis algorithm that would help select the best suitable miRNAs that target 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. 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. Expression of these miRNAs was tested in the brain tissue of 40 suicide completers and controls, and hsa-miR-4516 and hsa-miR-381-3p showed a trend for statistical significance. We also checked the expression of the target genes of these miRNAs, and for NR3C1 expression was lower in suicide completers compared to controls, which is in accordance with the available literature results. To determine the miRNAs that are most suitable for further suicidality research, more studies, combining in silico analysis and wet lab experiments, should be performed

Dynamic intercell communication between glioblastoma and microenvironment through extracellular vesicles

Glioblastoma is simultaneously the most common and most aggressive primary brain tumor in the central nervous system, with poor patient survival and scarce treatment options. Most primary glioblastomas reoccur and evolve radio- and chemoresistant properties which make them resistant to further treatments. Based on gene mutations and expression profiles, glioblastoma is relatively well classifiedhowever, research shows that there is more to glioblastoma biology than that defined solely by its genetic component. Specifically, the overall malignancy of the tumor is also influenced by the dynamic communication to its immediate and distant environment, as important messengers to neighboring cells in the tumor microenvironment extracellular vesicles (EVs) have been identified. EVs and their cargo can modulate the immune microenvironment and other physiological processes, and can interact with the host immune system. They are involved in tumor cell survival and metabolism, tumor initiation, progression, and therapy resistance. However, on the other hand EVs are thought to become an effective treatment alternative, since they can cross the blood–brain barrier, are able of specific cell-targeting and can be loaded with various therapeutic molecules

Epigenetics of Alzheimer\u27s disease

There are currently no validated biomarkers which can be used to accurately diagnose Alzheimer\u27s disease (AD) or to distinguish it from other dementia-causing neuropathologies. Moreover, to date, only symptomatic treatments exist for this progressive neurodegenerative disorder. In the search for new, more reliable biomarkers and potential therapeutic options, epigenetic modifications have emerged as important players in the pathogenesis of AD. The aim of the article was to provide a brief overview of the current knowledge regarding the role of epigenetics (including mitoepigenetics) in AD, and the possibility of applying these advances for future AD therapy. Extensive research has suggested an important role of DNA methylation and hydroxymethylation, histone posttranslational modifications, and non-coding RNA regulation (with the emphasis on microRNAs) in the course and development of AD. Recent studies also indicated mitochondrial DNA (mtDNA) as an interesting biomarker of AD, since dysfunctions in the mitochondria and lower mtDNA copy number have been associated with AD pathophysiology. The current evidence suggests that epigenetic changes can be successfully detected, not only in the central nervous system, but also in the cerebrospinal fluid and on the periphery, contributing further to their potential as both biomarkers and therapeutic targets in AD

DNA Methylation Patterns in Relation to Acute Severity and Duration of Anxiety and Depression

Depression and anxiety are common mental disorders that often occur together. Stress is an important risk factor for both disorders, affecting pathophysiological processes through epigenetic changes that mediate gene–environment interactions. In this study, we explored two proposed models about the dynamic nature of DNA methylation in anxiety and depression: a stable change, in which DNA methylation accumulates over time as a function of the duration of clinical symptoms of anxiety and depression, or a flexible change, in which DNA methylation correlates with the acute severity of clinical symptoms. Symptom severity was assessed using clinical questionnaires for anxiety and depression (BDI-II, IDS-C, and HAM-A), and the current episode and the total lifetime symptom duration was obtained from patients’ medical records. Peripheral blood DNA methylation levels were determined for the BDNF, COMT, and SLC6A4 genes. We found a significant negative correlation between COMT_1 amplicon methylation and acute symptom scores, with BDI-II (R(22) = 0.190, p = 0.033), IDS-C (R(22) = 0.199, p = 0.029), and HAM-A (R(22) = 0.231, p = 0.018) all showing a similar degree of correlation. Our results suggest that DNA methylation follows flexible dynamics, with methylation levels closely associated with acute clinical presentation rather than with the duration of anxiety and depression. These results provide important insights into the dynamic nature of DNA methylation in anxiety and affective disorders and contribute to our understanding of the complex interplay between stress, epigenetics, and individual phenotype

Difference in Methylation and Expression of Brain-Derived Neurotrophic Factor in Alzheimer&rsquo;s Disease and Mild Cognitive Impairment

Due to the increasing number of progressive dementias in the population, numerous studies are being conducted that seek to determine risk factors, biomarkers and pathological mechanisms that could help to differentiate between normal symptoms of aging, mild cognitive impairment (MCI) and dementia. The aim of this study was to investigate the possible association of levels of BDNF and COMT gene expression and methylation in peripheral blood cells with the development of Alzheimer&rsquo;s disease (AD). Our results revealed higher expression levels of BDNF (p &lt; 0.001) in MCI subjects compared to individuals diagnosed with AD. However, no difference in COMT gene expression (p = 0.366) was detected. DNA methylation of the CpG islands and other sequences with potential effects on gene expression regulation revealed just one region (BDNF_9) in the BDNF gene (p = 0.078) with marginally lower levels of methylation in the AD compared to MCI subjects. Here, we show that the level of BDNF expression in the periphery is decreased in subjects with AD compared to individuals with MCI. The combined results from the gene expression analysis and DNA methylation analysis point to the potential of BDNF as a marker that could help distinguish between MCI and AD patients

Difference in Methylation and Expression of Brain-Derived Neurotrophic Factor in Alzheimer’s Disease and Mild Cognitive Impairment

Due to the increasing number of progressive dementias in the population, numerous studies are being conducted that seek to determine risk factors, biomarkers and pathological mechanisms that could help to differentiate between normal symptoms of aging, mild cognitive impairment (MCI) and dementia. The aim of this study was to investigate the possible association of levels of BDNF and COMT gene expression and methylation in peripheral blood cells with the development of Alzheimer’s disease (AD). Our results revealed higher expression levels of BDNF (p COMT gene expression (p = 0.366) was detected. DNA methylation of the CpG islands and other sequences with potential effects on gene expression regulation revealed just one region (BDNF_9) in the BDNF gene (p = 0.078) with marginally lower levels of methylation in the AD compared to MCI subjects. Here, we show that the level of BDNF expression in the periphery is decreased in subjects with AD compared to individuals with MCI. The combined results from the gene expression analysis and DNA methylation analysis point to the potential of BDNF as a marker that could help distinguish between MCI and AD patients