Določanje metilacije DNA gena SLC6A4 pri žrtvah samomora s tehnologijo sekvenciranja naslednje generacije

Pucihar, Natalija (author), Videtič Paska, Alja (mentor)

Človeško vedenje oblikujejo genetski in okoljski dejavniki, ki vplivajo na pojavnost duševnih motenj in tudi samomorilnega vedenja. Znano je, da motnje v serotonergičnem sistemu lahko pomembno prispevajo k razvoju duševnih motenj, agresivnemu in impulzivnemu vedenju ter samomorilnemu vedenju. Številni raziskovalci so v svojih študijah pokazali, da imajo epigenetski mehanizmi, ki vplivajo na izražanje genov, potencialno vlogo pri samomorilnem vedenju. V nalogi smo preučevali stopnjo metilacije, ki je ena izmed epigenetskih modifikacij, gena SLC6A4, ki zapisuje za serotoninski transporter. Iz možganskih področij amigdale, insule, Brodmannovega področja 46 in hipokampusa ter krvi žrtev samomora in kontrolnih oseb smo izolirali DNA. To smo bisulfitno pretvorili in pripravili knjižnico tako, da smo z uporabo verižne reakcije s polimerazo (PCR) pomnožili izbrane dele otočka CpG kandidatnega gena ter del v njegovi bližini. Po PCR smo ustreznost pomnožkov preverili z agarozno gelsko elektroforezo in pomnožke očistili s paramagnetnimi kroglicami. Z uporabo tehnologije sekvenciranja naslednje generacije in statističnimi pristopi smo določili razliko v metilaciji posameznih citozinov pri žrtvah samomora glede na kontrolno skupino. Rezultati statistične obdelave metiliranih citozinov pomnožka SLC6A4_1 so pokazale, da je število statistično pomembnih metiliranih citozinov pri vseh štirih možganskih področjih in v krvi dokaj podobno. Stopnja metilacije pri žrtvah samomora glede na kontrolno skupino je bila v Brodmannovem področju 46, hipokampusu in v krvi znižana, v amigdali dokaj enaka, v insuli pa smo opazili povišano število metiliranih citozinov. Pri pomnožku SLC6A4_2 smo opazili, da je število statistično pomembnih metiliranih citozinov med preiskovanima skupinama oseb različno. V amigdali in insuli ni bilo opaženih statistično pomembnih metiliranih citozinov, v Brodmannovem področju 46 je bila stopnja metilacije citozinov pri žrtvah samomora glede na kontrolno skupino znižana, v hipokampusu pa zvišana. V krvi je vzorec metilacije mešan. Hipotezo, da obstajajo statistično pomembne razlike v stopnji metilacije DNA SLC6A4 med možganskimi področji in v krvi skupine žrtev samomora in kontrolne skupine smo potrdili, vendar pa so te razlike sorazmerno nizke.

• Language: Slovenian
• Keywords: samomor, epigenetika, metilacija DNA, SLC6A4, NGS
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2021
• PID: 20.500.12556/RUL-124858
• COBISS.SI-ID: 53186307
• Publication date in RUL: 24.02.2021

Secondary language

• Language: English
• Title: DNA methylation of SLC6A4 gene in suicide victims with next generation sequencing

Abstract

Human behavior is regulated by genetic and environmental factors, which impact on increased frequency of psychiatric behaviors and also suicidal behavior. It has been shown that changes in serotonergic system can have an important role in development of mental disorders, aggressive and impulsive behavior or suicidal behavior. Many researchers have shown that epigenetic mechanisms that influence the gene expression could have a potential role in suicidal behavior. In this thesis we have studied the DNA methylation level, which is one of the epigenetic modifications, of a gene SLC6A4, which encodes serotonin transporter. We have isolated the DNA out of brain tissue of amygdala, insula, Brodmann area 46, hippocampus and out of blood of suicide victims and control group. Afterwards we did the bisulfite conversion of the DNA and prepared a library by using a polymerase chain reaction ( PCR) to amplify a part of a CpG island of candidate gene and a part nearby. After PCR we checked the amplicons with agarose gel electrophoresis and cleaned the products using magnetic beads. With the use of next generation sequencing technology and statistical approaches we have managed to determine level of methylation of the DNA amplicons. After statistical analysis of the methylated cytosines of amplicon SLC6A4_1 we determined that the number of statistically significant methylated cytosines is fairly equal in all four brain areas and blood. Suicide victims had decreased methylation levels in Brodmann area 46, hippocampus and blood in comparison with control group. Methylation levels were mixed in amygdala and increased in insula. In DNA amplicon SLC6A4_2 differences in the number of methylated cytosines were noticed. In amygdala and insula there was no statistically relevant differentially methylated cytosine observed. The methylation level decreased in Brodmann area 46 of suicide victims in comparison with the control group and increased in hippocampus. In blood the methylation pattern was mixed. We confirmed the hypothesis that there are statistically relevant differences in methylation levels of DNA SLC6A4 among brain areas and blood of suicide victims and control group, however these differences are rather small.

• Keywords: suicide, epigenetics, DNA methylation, SLC6A4, NGS