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Browsing by Subject "DNA methylation"

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  • Laukkanen, Ida (2024)
    Epigenetics is the study of changes in gene expression without alterations in the DNA sequence. Epigenetic modifications, of which DNA methylation (DNAm) is the most known, are crucial for many biological events, especially for normal development and genomic imprinting. In imprinted genes, only one of the parental alleles is consistently monoallelically expressed. The epigenome can be altered by various environmental factors such as diet and chemicals. Moreover, evidence indicates that assisted reproductive technology (ART) is associated with distinct DNAm patterns. Still, it is unclear whether these alterations are a consequence of ART procedures themselves or the underlying subfertility. This thesis aimed to study whether ART procedures and subfertility are associated with aberrant DNAm at the imprinted DLK1-DIO3 locus in the human placenta. The genes encoded from this paternally imprinted locus are essential in mammals' fetal and placental development. Moreover, recent evidence suggests that downregulation of the DLK1 gene is associated with ART and subfertility. Therefore, two gene regions, the IG-DMR and the DLK1 promoter, were chosen as study objects. The IG-DMR acts as the main regulator of gene expression at the whole DLK1-DIO3 locus, and promoter regions are considered important regulators of the expression of their corresponding genes. Eight ART, four subfertility, and six control samples of human placental DNA were studied. The subfertility group consisted of couples who were committed to initiating fertility treatments but eventually got pregnant spontaneously. The study was performed using traditional bisulfite sequencing, after which the differences in DNAm levels between study groups were statistically analyzed. As expected, significantly decreased DNAm level was observed in the placentas of subfertile couples compared to controls. Surprisingly, no significant differences were addressed between ART and control groups in either region. The partly unexpected results are explained by the fact that aberrant methylation at distinct imprinted DMRs, including the IG-DMR, is caused by the in vitro culture media. Moreover, the results indicate that the regulation of DLK1 expression is more complicated than that solely by the IG-DMR and the DLK1 promoter. Further research should be dedicated to differentiating the impacts of ART and subfertility on the epigenome and phenotype to better understand the health implications of ART.
  • Akhondzadeh, Soheila (2016)
    Background: Epithelial ovarian cancer is the most common type of ovarian cancer and is the most lethal gynecologic cancer due to its late diagnosis. Compared to ovarian cancer, endometrial carcinoma, as the most common gynecologic malignancy, is referred to as the “curable cancer”, as it can be detected early. As aberrant promoter methylation patterns are a common change in human cancer, detection of promoter methylation status may help in early diagnosis. In this study, we used a custom-designed methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay as a rapid and easy method, to simultaneously detect the methylation status of multiple genes in ovarian and endometrial cancer samples. Aims: To design and test an MS-MLPA assay for analyzing promoter methylation of four genes associated with ovarian and endometrial cancers. The selected genes were HNF1 homeobox B (HNF1β), Ten-eleven translocation 1(TET1), L1 cell adhesion molecule (L1CAM), and AT-rich interactive domain 1A (ARID1A). These genes are known to have expression changes by DNA methylation. Methods: The promoter DNA methylation patterns of these four genes were analyzed in 15 cancer cell lines and 5 normal cell lines and DNAs using bisulfite sequencing. Six synthetic probe pairs were designed and optimized by applying them to cancer and normal cell lines and normal DNAs and comparing the results with those of bisulfite sequencing. Finally, the MS-MLPA assay was performed on patient specimens according to the MRC-HOLLAND MS-MLPA general protocol and methylation frequencies were calculated from MS-MLPA data. Results and conclusion: The MS-MLPA assay gave accurate methylation results with the 170 samples assayed. The HNF1B, L1CAM, and TET1 Genes were observed methylated in tumor samples whereas they were not methylated in the normal samples or showed very little methylation, suggested to be favorable diagnostic markers. MS-MLPA robustly and sensitively detects the promoter DNA methylation status.
  • Ukwattage, Sanjeevi (2019)
    Background- Colorectal cancer (CRC) is the third most common epithelial carcinoma. There is an increased risk of colorectal cancer in people with longstanding inflammation in the large intestine, including individuals with ulcerative colitis (UC). Epigenetic changes in CRC such as aberrant DNA methylation alterations are common changes in human cancer. The aim of this study is to identify the DNA methylation alterations of selected inflammation related genes in UC-CRC vs. Lynch syndrome (LS). Method- DNA was extracted from archival tissue specimens from normal and tumor samples from UC-CRC (n= 31), and LS-CRC (n=29). Methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) assays were used to detect CIMP status and CpG promoter methylation status of seven inflammation related genes. Microsatellite instability analysis was carried out using two mononucleotide repeat markers BAT25 and BAT26. Results- Increased hypermethylation frequencies in carcinoma vs. normal colonic mucosa were detected for all the inflammatory marker genes in specimens of UC-CRC patients. Statistically significant differences for methylation frequencies were observed in the NTSR1 gene (p value =0.008) and SOCS2 gene (p value =0.04) in specimens of UC-CRC patients. NTSR1 gene showed significantly increased methylation of normal colonic mucosae from UC-CRC vs. LS patients (p value=0.01). Conclusion- UC-CRC and LS tumor specimens revealed varying frequencies of hypermethylation in all the inflammatory genes. Methylation of the NTSR1 in the normal colonic mucosa suggests a possible field defect in UC-CRC, and could thus be used as an early biomarker to detect increased UC-CRC risk in non-neoplastic epithelium.
  • elDandashi, Rahaf (2021)
    Epigenetics is the study of changes in gene function without affecting the DNA sequence. Epigenetics studies the effects of the environment and behavior on the genome. Researchers have been able to detect several epigenetic modifications such as –DNA methylation, histone acetylation, and microRNA-associated gene silencing. Changes in the epigenome are essential for proper cell function and normal development and can also be induced by environmental factors. Stress is defined as a biological response to physiological and psychological demands which can affect cellular homeostasis. Factors such as prenatal life stress can affect gene function without directly altering the DNA nucleotide sequence. Elevated levels of stress can immobilize with the ability to impair cognitive function. There is evidence that suggests the involvement of epigenetic regulation in disorders such as addiction, depression, schizophrenia, and cognitive dysfunction. Therefore, this systematic review discusses recent findings of the role of epigenetics in prenatal exposure to stress. To achieve this, the thesis will cover different subtopics from genetics, neurobiology, and diseases, neuroscience, biological psychiatry, life sciences, medicine, behavioral brain research, biochemistry & molecular biology, as well as neuroendocrinology. Research questions are 1) Is there an association between epigenetics and prenatal stress? 2) What kind of mechanisms have been found? 3) What kind of techniques have been used in the identification of potential epigenetic mechanisms? What genes are associated with these epigenetic changes?. This study followed the "The Preferred Reporting Items for Systematic Reviews and Meta-Analyses" (PRISMA) guideline checklist. Eligibility criteria and search terms where be selected and documented to offer the widest range of articles covering the subjects of this study. A literature search was done using PubMed/Medline, Google scholar, and gray literature. The last sample comprised 59 articles. Data were extracted so that the participants, intervention, comparisons, and outcomes were included. The literature search conducted in this systematic review identified a few findings. First is that the majority of animal and human studies found a significant or moderate association between epigenetics and prenatal stress. Second, DNA methylation is the most studied epigenetic mechanism in maternal exposure to stress Third, genome-wide studies were more common in human studies than in animals and the most widely used method used is Infinium HumanMethylation450 Bead Chip. However, the common methods used in human and animal studies are most likely because of the small sample size and causation cannot be determined. Finally, NR3C1 and FKBP5 genes were the most studied in human studies where they showed the strongest association between prenatal stress and epigenetic modifications. While in animal studies, the most studied genes were Bdnf and Dnmt1 as they showed a significant methylation level after maternal prenatal stress exposure. In conclusion, maternal prenatal stress could trigger epigenetic alterations in neonates in both animals and humans. This holistic review detailed and evaluated locus-specific and studies exploring current knowledge about associations between maternal prenatal stress and epigenetic changes.
  • Koivumaa, Minna (2020)
    Tiivistelmä – Referat – Abstract Ewing sarcoma is a rare bone and soft tissues cancer that occurs mainly among children and young adults. It is an aggressive cancer. Treatment of Ewing Sarcoma Family of Tumours (ESFT) primarily includes surgery, radiation and chemotherapy. The treatment protocol depends on the presence of tumour metastases at the time of diagnosis. In the treatment of local tumours, the 5-year patient survival rate has increased from 50% to 70%. However, patients that have tumour metastases at the time of the diagnosis or have a recurrent disease, the five-year survival rate is only 25%. As the current treatment options have reached their limits, it is important to develop more advanced therapies. DNA methylation is an epigenetic event that affects gene expression. By comparing the methylation level of the DNA in gene promoter regions in ESFT cancer cells to the methylation level of DNA in gene promoter regions in normal cells it could be possible to discover genes and signalling pathways that are important in the development of ESFT and that could be potential drug target molecules. The aim of this study is to find out the genome-wide gene promoter DNA methylation status in Ewing sarcoma cell line samples and Ewing sarcoma patient tumour samples compared to a normal reference sample. Another aim is to find gene promoter regions that are differentially methylated in the Ewing sarcoma cell line samples and the Ewing sarcoma patient tumour samples compared to the normal reference sample. Materials and Methods The Ewing Sarcoma cell line samples (12) were obtained from the Laboratory of Oncologi Research, Instituti Ortopedici Rizzoli Laboratory, Bologna, Italy. The Ewing sarcoma patient tumour samples were pre-isolated DNA samples already in Finland. The normal reference sample was a commercial mesenchymal cell line sample. From the Ewing Sarcoma cell line samples and the normal reference sample, DNA isolation was done by using phenol-chloroform method. DNA methylation profiling of the samples was performed by combining MeDIP (methylated DNA immunoprecipitation) protocol with 2-set promoter microarray hybridization protocol provided by Agilent Tecnologies company. DNA methylation data that was received from the microarrays was normalized and pre-processed with the Feature Extraction software provided also by the Agilent Technologies company. Visualization of the DNA methylation data was performed by using Chipster analysis software provided by CSC. To measure the level of DNA methylation at the gene promoter regions, a log2ratio value was calculated for every gene promoter region in all the sample types. To find gene promoter regions that were differently methylated, a log2 fold change value was calculated from the log2ratio values between the Ewing Sarcoma cell line cancer samples and the normal reference sample and between the Ewing sarcoma patient tumor samples and the normal reference sample for each gene promoter region. The log2 fold change value was also calculated between the Ewing Sarcoma cell line cancer samples and the Ewing Sarcoma patient tumor samples. After this a t-test was performed to determine the statistical significance of the log2 fold change values. Detection of genome-wide DNA methylation levels at the gene promoter regions in the Ewing sarcoma cell line samples and the Ewing sarcoma patient tumour samples compared to the normal reference sample was performed by averaging log2 fold change values. The same calculation method was used to detect the differences in the genome-wide DNA methylation levels at the gene promoter regions between the Ewing sarcoma cell lines and the Ewing Sarcoma patient tumour samples. Results Differences in the DNA methylation levels at the gene promoter regions were detected between the Ewing Sarcoma cell line samples, patient tumour samples, and the normal reference sample. Genome-wide measurement of the DNA methylation levels at the gene promoter areas showed that the Ewing sarcoma cell lines had more DNA methylation at the gene promoter regions than the patient tumour samples and the normal reference sample. The patient tumour samples showed less DNA methylation at the gene promoter regions compared to the Ewing sarcoma cell lines and the normal reference sample. Differentially methylated gene promoter regions between the Ewing sarcoma cell lines and the reference sample were found 16. In the patient tumour samples, also 16 differently methylated gene promoter regions were found compared to the normal reference sample. Differentially methylated gene promoter regions between the Ewing sarcoma cell lines and the patient tumour samples were 56.
  • Puustinen, Taru (2024)
    Colorectal cancer (CRC) poses a considerable global health challenge, with high mortality rates despite advancements in cancer research. Approximately one in ten CRC cases have a hereditary basis, with Lynch syndrome and Familial adenomatous polyposis standing as the two predominant cancer-predisposing syndromes. These conditions are mainly attributed to specific dominant germline mutations in CRC-related genes. However, as the role of epigenetic modifications, including DNA methylation, has become increasingly recognized in colorectal tumorigenesis, identifying these distinct signatures is paramount in gaining deeper insights into the molecular mechanisms underlying CRC. This thesis, conducted as a part of ongoing research into the DNA methylation profile of Lynch syndrome and Familial adenomatous polyposis-associated colorectal tumors, aimed to validate methylation patterns previously obtained from a genome-wide Illumina Methylation EPIC BeadChip array. The validation was performed by utilizing direct bisulfite sequencing across 12 selected gene regions on CRC cell lines and histologically normal control samples and comparing the methylation status to the EPIC array results. The genomic regions selected for this analysis were chosen based on differential methylation observed with EPIC and literature and were limited to regions that included at least one GCGC site for the HhaI digestion enzyme for future in-house MS-MLPA probe design. The results of this study demonstrate a clear differential methylation pattern of the selected genes, with CRC cell lines generally exhibiting higher methylation levels compared to non-cancerous samples, as expected. Specifically, high concordance between EPIC results and bisulfite sequencing data was observed in the methylation status of ADHFE1, EYA4, ITGA4, FBLIM1, and SEPT9, whose connection to CRC has been also shown in the previous studies. Further investigations of the genes in this study could contribute to a better understanding of epigenetic changes underlying colorectal tumors and hold the potential for developing novel biomarkers for early diagnosis and improved patient prognosis.