DNA methylation of candidate genes and quantification of cfDNA in patients with depressionAtanasova, Marija (Avtor) Videtič Paska, Alja (Mentor) depressionDNA methylationBDNFCOMTSLC6A4cell-free DNADepression is a common and heterogeneous mental disorder. When a patient, diagnosed with depression, does not achieve an adequate response to treatment with at least two antidepressants, it is considered that patient has developed a treatment-resistant depression (TRD). As an alternative treatment for TRD, repetitive transcranial magnetic stimulation (rTMS) has been proposed. Family, twin and adoption studies have emphasized the role of genetic factors in depression. Genes coding for brain-derived neurotrophic factor (BDNF), catechol-O-methyltransferase (COMT), and solute carrier family 6 member 4 (SLC6A4) are of high interest for research regarding their association with different mental disorders. DNA hypermethylation is associated with gene silencing and it has been in the focus of epigenetics research, where it has been shown that epigenetic changes could play an important role in the development of different diseases including depression. cell-free DNA (cfDNA) in different body fluids has also been determined. Higher levels of cfDNA are often correlated with physiological and physical stress situations, cancer, and different mental disorders, such as schizophrenia. The experimental part of this master thesis consists of two parts: determination of the methylation of the candidate genes using the technology of next generation sequencing (NGS) and quantification of plasma cfDNA. Blood samples were taken of patients diagnosed with TRD who did undergo rTMS, before and after the treatment, and from a control group of patients with depression. We performed DNA isolation and bisulfite conversion on the isolated DNA. Polymerase chain reaction (PCR) was used for amplification of distinct CpG island parts of the genes of interest. Using the NGS technology and the statistical tools FastQC, TrimGalore, Bismark and R (methylKit annotatr) we have analysed the average amplicon methylation and the number of methylated cytosines in the assayed BDNF, COMT, and SLC6A4 CpG islands. In the analysis we compared three groups: TRD patients before the rTMS treatment, patients who underwent the rTMS treatment and the control group. Also, we performed cfDNA isolation in order to determine its concentration before and after rTMS treatment. The concentration was determined with droplet digital PCR. The results have shown that there is no significant difference in the methylation level of the candidate genes, nor in the cfDNA level. The samples did not differ considerably. It is difficult to derive a definitive conclusion since the number of samples was rather small as this was an exploratory study. There is a need for larger and well-defined cohorts in order to dissect the results further.20202020-02-06 13:15:01Magistrsko delo/naloga113841sl