Browsing by study line "Genetics and genomics"

Sex determination in humans occurs via the sex chromosomes, X and Y. Females carry two X chromosomes while males are XY individuals. Due to this X chromosome distribution the expression of X-linked genes is balanced with a process called X chromosome inactivation (XCI) where one of the X chromosomes is silenced, selected either randomly or preferentially, in early female embryogenesis. X-linked disorders are more prevalent in males as, generally, in females the effects of a disease-causing variant in other of the X chromosomes can be compensated with the normal allele on the other X whereas male express the allele on their only X chromosome. However, cases of heterozygous females manifesting an assumed recessive X-linked disorder have been reported although the symptoms are usually milder in these cases than in males. One suggested reason behind this is a skewed XCI where the majority of female’s cells express the mutated allele. The main goal of this thesis was to examine how often heterozygous female carriers have symptoms of X-linked disorders. To achieve this goal, likely pathogenic and pathogenic X-chromosomal variants were retrieved from the ClinVar database and their global allele frequencies were examined from The Genome Aggregation Database (gnomAD). The genetic and phenotypic data of 500,000 individuals from the UK Biobank (UKB) were used to conduct genetic association analyses between the ClinVar variants and quantitative traits related to their reported phenotypes. The associations were tested in males and in females separately to allow for examination of sex-specific effects and inheritance models via the comparison of effect sizes. 89 (likely) pathogenic variants were detected from UKB, and the majority of these were extremely rare with minor allele frequency below 0.01% in the global population. 11 and 27 of them were selected for the association analyses for the male and female populations of UKB, respectively, after filtering out variants that did not meet requirements such as enough carriers. One to five quantitative traits were chosen for each variant resulting in 28 tests among males and 87 among females. These analyses showed few significant associations while the majority of the tested variants were observed to have no effects on the chosen trait. The most statistically significant association was observed with variant rs137852591 on the gene AR (androgen receptor) in males. The variant was related to lower muscle mass and shorter height that are associated partial androgen insensitivity syndrome reported in ClinVar for this variant. Nominally significant associations were seen with this variant and the same traits in heterozygous females suggesting that there might be, indeed, symptoms of the syndrome in females as well. Additionally, in both sexes variants on gene G6PD seemed related to traits that are characteristics of glucose 6 phosphate dehydrogenase deficiency. The limitations of these databases must be taken into account when conducting studies utilizing them. However, this thesis demonstrated that heterozygous female carriers may have symptoms of X-linked disorders assumed to have recessive inheritance pattern. In the future, a wider set of phenotypes could be used to investigate the impacts of the X-linked variants more broadly.

Rare mutations in the primate specific ZNF808 gene are a novel cause of pancreatic agenesis, a congenital developmental disorder that leads to neonatal diabetes. ZNF808 loss-of-function has been shown to lead to aberrant activation of regulatory MER11 elements, followed by upregulation of genes in proximity to these elements and increased expression of hepatic lineage markers. These findings suggest ZNF808 to play a key role in balancing the differentiation of endoderm progenitor cells between pancreatic and liver lineages during early human development. This thesis work aimed to study the gene regulatory mechanisms of ZNF808 in the differentiating endoderm progenitor cells to understand its function in controlling pancreatic lineage specification. This was achieved by comparing the lineage specification processes in wild-type (H1) and ZNF808 knockout (H1-ZNF808-KO) human embryonic stem cells (hESCs) during pancreatic differentiation. Further characterization of cellular heterogeneity and gene expression profiles upon ZNF808 loss was done using single-cell RNA sequencing (scRNA-seq). To validate the role of ZNF808 as the mediator of the observed lineage specification bias, the phenotype rescue was examined in a ZNF808 knockout overexpression cell line (H1-ZNF808-KO-OX). The results of this study demonstrate a clear lineage specification bias in the ZNF808 knockout, seen as divergence of the multipotent endoderm progenitors towards alternate hepatic and biliary fates at the posterior foregut stage. By modifying the pancreatic differentiation protocol, we were able to observe phenotype manifestation and cellular heterogeneity suppressed in the standard differentiation conditions. The scRNA-seq data analysis revealed the emergence of a biliary cell population showing upregulation of several hepatic markers, suggesting an alternative lineage specification process governed by ZNF808. Additionally, preliminary results from ZNF808 overexpression showed rescue of the ZNF808 knockout phenotype, further supporting its critical role in the normal pancreatic lineage development. In conclusion, these findings demonstrate the important role of ZNF808 in early human pancreatic development and warrant further studies on the detailed gene regulatory network guiding pancreatic lineage specification.

Gastrointestinal stromal tumours (GISTs) are rare mesenchymal neoplasms that arise from the interstitial cells of Cajal, the so-called pacemaker cells of the intestine. GISTs typically contain an oncogenic driver mutation either in the proto-oncogene KIT or platelet-derived growth factor receptor A (PDGFRA), which belong to the class III receptor tyrosine kinases. Patients with a high-risk or advanced disease are standardly treated with a tyrosine kinase inhibitor imatinib. Despite this molecularly targeted treatment, many patients experience disease relapse, after which the prognosis is poor. Personalised treatment is rarely offered to patients as a first-line treatment option, even though several targeted therapies have been approved for GIST. Increasing treatment personalisation could improve treatment outcomes, yet the lack of patient-specific research models for GIST hinders the research. Three-dimensional (3D) cell culture models are widely used in cancer research to study the molecular mechanisms underlying tumorigenesis. Their ability to mimic the tumour biology and microenvironment is superior compared to the traditional two-dimensional (2D) cell culture model. For several cancers, these cell culture models have also been researched as platforms for personalised treatment selection with promising results. This thesis project aimed to study UPM Biomedicals’ GrowDex-based 3D cell culture model as a potential platform for personalised treatment selection for GIST patients. GrowDex is a plant-derived hydrogel that resembles the extracellular matrix. Another aim of this project was to set up a Sanger sequencing protocol covering frequently mutated areas in GIST to facilitate the validation of this cell culture model through drug testing on patient samples. To assess the GrowDex microenvironment, the viability and proliferation of two GIST cell lines, GIST-T1 and GIST48 were monitored. Furthermore, the imatinib response of GIST-T1 in GrowDex was assessed and compared to the response in other cell culturing conditions. The Sanger sequencing protocol was optimised using the aforementioned cell lines and then applied to GIST patient samples. The results of this project demonstrated that GrowDex provides a suitable microenvironment for culturing GIST cells and supports their 3D growth. GIST-T1 cells were less sensitive to imatinib when cultured in GrowDex in comparison to the 2D culturing condition, which is likely explained by the 3D organisation of the cells. Finally, the Sanger sequencing protocol was used to uncover the KIT/PDGFRA mutation status of several GIST patient samples. In conclusion, these results provide important information for further development of this cell culture model with patient samples.

Breast cancer is the most common cancer in the world and among women the most cancer deaths causing cancer. MYC is a proto-oncogene, which becomes oncogenic when its expression is deregulated in cancer. MYC is commonly overexpressed in human tumours and this alteration is associated with aggressive cancer phenotype. Furthermore, alterations in the MYC network have been found in the great majority of breast cancers. MYC promotes mitochondrial apoptosis causing a cancer vulnerability, however, in cancer cells the apoptosis is often prevented by antiapoptotic BCL-2 family members. In this study, cell viability and cell death analysis of treated triple-negative breast cancer cell lines together with dendritic cell activation experiments were conducted. This study aimed to find the most potent BCL-2 family antagonist (BH3 mimetic) to combine with metformin to overcome the antiapoptotic BCL-2 family proteins inhibition of MYC-induced apoptosis. In addition, this study determined whether the combinations could induce immunogenic cell death to further intensify cancer cell killing through anti-tumour immunity. In this study, BH3 mimetics combined with metformin were found to induce cell death and reduce cell viability in TNBC cell lines. In addition, metformin and BH3 mimetics were found to activate dendritic cells directly and through immunogenic cell death of cancer cells. However, no MYC-dependent cell death or immunogenic cell death were observed, and this study was unable to indicate the most potent BH3 mimetic to combine with metformin.