Browsing by master's degree program "Magisterprogrammet i genetik och molekylära biovetenskaper"

Exercise-induced hyperinsulinism (EIHI) is a pathological condition characterized by aberrant insulin secretion triggered by physical exercise or pyruvate exposure. The monocarboxylate transporter protein (MCT1), encoded by SLC16A1, is ubiquitously expressed in almost all cell types except pancreatic islet cells. In patients with EIHI, mutations in the regulatory regions of the SLC16A1 gene are thought to lead to the unwanted expression of MCT1 on the beta cell membrane, allowing the influx of elevated lactate and pyruvate blood levels during exercise. These substrates feed into the Krebs cycle, increasing insulin release. This excessive insulin secretion can lead to hypoglycemia during exercise, causing weakness, syncope, and confusion. Since EIHI has never been studied using a human stem cell-derived islets, this thesis aims to establish a robust model in which to investigate the disease mechanism in detail. To achieve this, we reprogrammed EIHI patients’ fibroblasts into a stable pluripotent state and further differentiated them into functional pancreatic stem cell-derived islets (SC-islets) in vitro. These SC-islets were then matured further in vivo (in immunocompromised mice) and compared to healthy SC-islet controls. Rigorous quality control measures were implemented throughout the differentiation process to ensure its efficacy and the expression regulation of SLC16A1 was studied during SC-islet development. Extensive phenotypic characterization was conducted using immunohistochemistry, quantitative gene expression level analysis, and insulin secretion assays with glucose and pyruvate. Contrary to expectations, the results of this study demonstrated that despite the SLC16A1 promoter mutation, the expression of SLC16A1 was downregulated similarly to the control cell line during development in vitro, resulting in similar pyruvate-stimulated insulin secretion to the control cells. Interestingly, immunohistochemical analysis of in vivo implanted SC-islets showed a clear phenotype with an increased number of MCT1-positive cells only in the mutant grafts, some of which were endocrine cells. In conclusion, the phenotypic manifestations of EIHI were not visible in the setting of in vitro modeling, which was attributed to the similar expression levels of MCT1 in both the mutant and control cell lines. However, following in vivo implantation, there was a noticeable increase in MCT1 expression exclusively in the mutant cells. This finding suggests a distinct regulatory mechanism of MCT1 expression, which might be impacted by the in vivo surroundings and the maturation state of human islets.

The endoplasmic reticulum (ER) is an important organelle of the cell where a high number of proteins are synthesized and modified to obtain their final structure. Therefore, the ER stress, which is caused by accumulation of unfolded proteins in the ER, is not to be taken lightly since it could contribute to many diseases, such as cancer and neurodegenerative diseases. The response to the ER stress is the unfolded protein response (UPR), which is an adaptive system that helps in adjusting for increased folding needs within the ER. One of the main protein branches in the UPR is inositol requiring enzyme 1 (IRE1). IRE1 detects the status of protein folding inside the ER and initiates the UPR signaling pathway to achieve either normal folding status or cell death. The aim of this research was to express yeast IRE1 in E.coli and human IRE1 in insect cells, purify with affinity chromatography and study the IRE1’s crystal structure with a small molecule modulator that could possibly enhance its activity. The protein was expressed successfully and purified with glutathione S-transferase (GST) tag, and the activity of the pure protein was determined. The structural studies were not fully completed since the absolute purity and yield that was necessary for crystallization was not achieved due to loss of protein during gel filtration and precipitation. Based on the results it is likely that the structure of the protein could be solved and further biochemical and structural studies with F10 are possible.

Lynch syndrome (LS) is the most common cancer predisposition disease caused by dominantly inherited pathogenic variant (PV) of a mismatch repair (MMR) gene leading to a defective gene allele. The four major MMR genes encode MMR proteins – MSH2, MSH6, MLH1 ja PMS2 – that participate in the proofreading and repairing of the daughter strand for mismatches after every replication. The inherited PVs predispose to cancer development as only one somatic allele loss is required for biallelic loss according to the Knudson’s “two-hit” hypothesis. The biallelic loss of an MMR-gene leads to disrupted protein function altering the MMR process. When mismatches are left unrepaired, genomic instability is caused, which can eventually lead to tumorigenesis. Especially, the risk of colorectal cancer (CRC) and endometrial cancer (EC) is increased in LS. The predisposition syndrome, LS, is important to detect as early as possible to decrease the risk of cancer by prevention and surveillance. The MMR genes and their defects vary in their consequences to the repair process considerably, and thus, it is crucial to know the different characteristics and functional effects of them when estimating the level of cancer risk. Variants of uncertain significance (VUS) are especially prevalent among LS variants. More information about their impact to the disease can be acquired by in vitro and in silico methods, for instance. The main goal of the efforts for early detection and prevention is to reduce cancer morbidity and mortality. In this thesis, the pathogenicities of MSH2 and MSH6 variants were studied with DiagMMR assay, which has been developed for studying the protein function of these genes. In addition to the traditional agarose gel electrophoresis (AGE), the samples were also analyzed by a fragment analyzer, Labchip, that bases its function on capillary electrophoresis. This way the MMR detection efficiency of the methods could be compared. Samples were collected as skin biopsies from controls and LS patients with known MMR gene variants by Helsinki University Central Hospital (HUCH). InSiGHT database, that collects the different MMR-gene variants and their pathogenicity classification, was used to ensure that different kinds of variations, both pathogenic (class 5) and currently internationally unlisted variants, were analysed. The skin samples were cultured to acquire primary fibroblasts for nuclear protein extraction. The level of pathogenicity was revealed by MMR-protein activity when substrate DNA with a mismatch was added to the extract. Then, restriction enzymes were used for producing fragments of different lengths, depending on the repair action, and the MMR efficiency was visualized by both electrophoretic methods. Additionally, MAPP-MMR tool was used for studying the MSH2 mismatch variants in silico. By comparing the results from these two methods, we show that the more quantitative Labchip brings diagnostic value to DiagMMR suggesting 100% specificity (n=10) and 90,9% (n=11) sensitivity in reference to the variant information. For example, MSH6 c.3103C>T, which is listed as pathogenic in InSiGHT, was more consistent in giving a MMR deficient (dMMR) result with Labchip. Difference in the functional detection could be seen particularly with the MSH6 variants, but the differences were less notable when Labchip results were compared to the previous interpretations of the samples made based on the validated DiagMMR protocol. With the unlisted MSH6 variants, c.3139dupT was detected as dMMR by Labchip which was in unison with the previous interpretation. Another one, MSH6 c.551delA, was seen as MMR proficient (pMMR) in all the results by both the methods, and with the previous interpretation being unclear, which highlights the importance of further testing of this variant. There was also one unlisted variant (c.1805T>C) among MSH2 for which we got uniform dMMR results in two patients. The high MAPP-MMR score (25.150) for the MSH2 p.Leu602Pro amino acid change also supported the evidence gained of the pathogenic nature of this variant. As a conclusion, DiagMMR can be used reliably for MMR efficiency analysis, especially when performed together with a more quantitative analysis method.

Lifespan is a key fitness trait, together with fecundity, dispersal, and growth. In addition to environmental factors shaping variation in lifespan, it is also influenced by genetic components. Based on theory, genetic variation in lifespan is expected to be reduced due to its high relevance to fitness. However, due to trade-offs between different life-history traits and the variable or unstable environmental conditions organisms face in nature, life-history traits are also expected to sustain higher genetic variation. From studies in model organisms, such as the fruit fly and the roundworm, researchers have uncovered key insights into the genetic basis of lifespan. Some genes have been shown to contribute more to lifespan than others and different species seem to share homologous genes influencing lifespan that have been conserved. Many of these genes relate to the insulin receptors and insulin signaling processes. The allelic variation and over- or under-expression of these genes have been shown to be associated with changes in lifespan. However, regardless of our accumulating knowledge of these genes in impacting lifespan under laboratory conditions, we have little understanding of the role of these genes impacting variation in lifespan under more natural conditions. In general, assessment of genes affecting variation in lifespan in natural populations is rare, even under circumstances where we know that the lifespan has a heritable component. The Glanville fritillary (Melitaea cinxia) is a butterfly that inhabits most of Europe. It is used as a model species in ecology and evolution in relation to metapopulation dynamics and spatially structured habitats. It has been studied extensively both under experimental conditions and via observational studies in the field. The Glanville fritillary butterfly works as a good model organism for assessments of genetic components of life-history variation, as vast amounts of genomic and ecological data are already available. In this thesis, I aim to shed light on the genetic background of lifespan by using the Glanville fritillary as a model organism. More specifically, I will test the association of some well-known lifespan-related candidate genes with a phenotypic variation on the butterfly’s adult lifespan based on previously obtained experimental data on individuals collected from the natural metapopulation during the larval stage.

The European rabbit (Oryctolagus cuniculus) is a small mammal native to the Iberian Peninsula, but introduced by humans to all continents except Antarctica. The rabbit has been a remarkably successful invasive species due to its generalist nature and fast reproduction. Its spreading has mostly been destructive to the local nature, and humans have used fatal rabbit diseases such as rabbit haemorrhagic disease (RHD) to control harmful populations. The rabbit population in Helsinki is one of the most northern annually surviving rabbit populations in the world. It is believed to have originated from escaped pet rabbits in the late 1980s, and in the early 2000s, the rabbits spread rapidly around the Helsinki area. RHD spread unintentionally to Finland in 2016, and the disease caused a significant reduction in the Helsinki rabbit population. Rabbit population genetics has previously been studied in several countries, but never before in Finland. The aim of the thesis was to examine the genetic diversity and population structure of the Helsinki rabbit population before and after the RHD epidemic, and to compare the results to similar preceding rabbit population genetic studies. Rabbit populations have previously been found to recover from major population crashes without a notable loss in genetic diversity using DNA microsatellite markers. The recent RHD epidemic in Helsinki provided an opportunity to study, whether a rabbit population can recover from a population crash even in a harsher environment without losing genetic diversity. To conduct genetic analysis, fourteen DNA microsatellite loci were genotyped from individuals caught during two distinct time periods, in 2008-2009 (n=130) and in 2019-2020 (n=59). Population structure was observed in both temporal rabbit populations with small but significant FST values. The 2019-2020 population was more diverse than the 2008-2009 population in terms of allele numbers and expected heterozygosity. This result was unexpected considering the recent RHD-epidemic but could be explained by gene flow from new escaped rabbits. Compared to other wild rabbit populations around the world, the Helsinki area rabbits exhibit significantly lower genetic diversity. Bottleneck tests showed a significant signal separately in both temporal populations, but the RHD bottleneck cannot be distinguished based on the tests. The results could be biased by new gene flow, or the initial bottleneck caused by the founder effect of only a few pet rabbits. The rabbits have demonstrated their adaptation and survival skills in the cold climate of Helsinki. The population has significantly lower genetic diversity compared to other wild populations, yet recovered from a major RHD epidemic without reduction in genetic diversity under these more extreme environmental conditions. It has been proven again; the rabbit is a thriving invasive species.

Preeclampsia is a vascular pregnancy disorder characterized by new-onset hypertension and proteinuria and/or new-onset preeclampsia associated symptoms during the second half of pregnancy. The pathophysiology of the disorder is not fully understood, but incomplete placentation and maternal tolerance towards fetal tissue are known to play a part in the disease pathogenesis. Predisposing factors include nulliparity, obesity, diabetes, chronic hypertension and autoimmune diseases. Furthermore, women who have experienced preeclampsia are more susceptible to cardiovascular disease later in life. One established biomarker for preeclampsia is the increased concentration of the soluble Fms-like tyrosine kinase 1 (sFlt1) in the maternal serum. sFlt1 is frequently overexpressed in preeclampsia and it is linked with angiogenic imbalance and endothelial dysfunction, although its role in the disorder is not completely clear. Preeclampsia has a genetic background. There are protective and predisposing variants in and near the Fms related tyrosine kinase 1 gene (FLT1; coding for sFlt1) that have been associated with preeclampsia either in the mother or in the fetus. In this study, five genetic polymorphisms over a 2.3 kb region in the 3’ untranslated region of FLT1 were genotyped by Sanger sequencing and fragment analysis in altogether 1200 individuals consisting of case and control mother–child pairs of the Finnish Genetics of Pre-eclampsia Consortium (FINNPEC) cohort. These polymorphisms were tested for association with various preeclampsia-related phenotypes by Fisher’s exact test. In the maternal genome, the minor alleles of rs17086497 and rs57760154 were associated with extreme hypertension (systolic blood pressure >180 mmHg) (p=0.004, OR=1.77) and obesity (p=0.023, OR=1.63). Homozygosity for these minor alleles was associated with pregnancy complications in general (p=0.026, OR=2.53) and the early-onset form of preeclampsia (p=0.004, OR=3.34). Additionally, the minor alleles of rs9554314, rs3138582 and rs149279513 were associated with extreme hypertension (p=0.045, OR=1.63) and obesity (p=0.023, OR=1.78). Moreover, a suggestive association to severe proteinuria (> 5 g/24h) was found in the maternal genome. In the fetal genome, significant negative associations were reached for rs17086497 and rs57760154 in terms of the serum concentration of sFlt1 in the preeclampsia group (p=0.008, OR=0.23). Overall, the results seem to link the studied region in the maternal genome to preeclampsia with severe features. This supports the idea of preeclampsia as a heterogeneous disorder with varying etiology and mechanisms and thus highlights the importance of differentiating between the various sub-phenotypes. For example, the association of the same allele in the fetal genome with lower maternal sFlt1 levels and in the maternal genome with severe symptoms of preeclampsia suggests that the sFlt1 level might not be a good measure in all patients. Additionally, the observed associations with extreme hypertension and obesity point to the possibility that this region might be relevant for the endothelial damage that is thought to be a central factor in creating the later-in-life disease susceptibility.

The intestinal stem cells (ISC) are responsible for the regeneration of the intestine epithelial barrier after acute injury and for the replenishment of its cells overall. How the ISC activation and resulting proliferation is controlled is complex and still under study. The ISCs of the midgut, which is the functional analogue to mammalian small intestine, are also highly responsive to changes in nutrition, and with proper methodologies it is possible to study the effects of diet on stem cell activation. The metabolic flux of the nutritional components of the diet can then shed light on which metabolic pathways are necessary for nutrient-dependent proliferation. One nutrient that has garnered interest is glutamine (Gln). It is well established that glutamine supplementation can in parenterally fed patients diminish intestinal barrier atrophy, extend the time the patient can be kept under the regime, and increase survivability of critically ill patients. Consequently, glutamine or its downstream metabolites may have stem cell activating characteristics. However, the exact regulatory mechanisms and specific effects of Gln are not well known, and studies have found contradictory results on the beneficial effects of Gln supplementation. Glutamine itself is a conditionally essential amino acid that has a variety of functions: it is an important source of nitrogen and cellular energy and contributes carbon into the tricarboxylic acid cycle (TCA) and is involved in protein and nucleotide synthesis. In this thesis, the effects of Gln supplementation on the cell populations of D. melanogaster were studied via microscopy and computational analysis. Cross-breeds of fruit fly were established to lineage label the ISC with a GAL4/UAS driver system. Confocal microscope was used to image the midguts which were then analysed with Imaris software. A novel analysis method was developed to study population changes and varying features of the cells in the midgut in an unprecedented region-by-region bulk analysis. Earlier studies into nutrient control of ISC have had limited focus within the midgut and might have consequently given a restricted view of ISC activation. This new Longitudinal Analysis of Midgut (LAM) can be utilized in a diverse set of further studies to describe conditional variation within midgut, and possibly other tissues. Gln was found to increase total cell numbers to comparable levels with well-fed midguts, and to drive limited endoreplication in enterocytes. Lineage labelled cell population grew primarily in the R3 and R4 regions of the midgut. Additionally, enteroendocrine cells (EE) were greatly increased in the posterior part of R3 but had conceivable minor increases along the whole length of the midgut. Improved nutrition was also found to affect the proportions of the midgut, presenting itself as elongated posterior and stunted anterior. Overall, the pipeline and analysis method established during this study enable more expeditious research of effects of other nutritional components and allows for study of effects of other mechanisms, for example how gene knock-downs or altered gene activities affect cell populations of the midgut.

The aim for this project is to set up a high-content imaging pipeline for phenotypic analysis of single cells in peripheral blood mononuclear cell (PBMC) samples from healthy blood donors. The blood donors selected for the optimization experiments are known to carry specific allele variants of interest, based on an earlier FinnGen study. The main question is whether these genetic differences result in phenotypic changes in the PBMCs that can be identified by microscopic imaging and AI-guided image analysis. In this Pro Gradu work, I have optimized the pipeline of PBMC sample handling, immunostaining, and phenotypic imaging. PBMCs were gathered from healthy donors at the Blood Service Biobank. The frozen PBMC samples were thawed, and cells were plated on 384-well plates prior to immediate fixation with paraformaldehyde. The cells were then stained with fluorescent cell markers based on the Cell Painting assay (Bray et.al. 2016), followed by wide-field and confocal imaging with Opera Phenix high-content confocal microscope (FIMM High Content Imaging and Analysis unit). Novel deep learning methods are now being developed (Pitkänen group) to automatically learn phenotypes from the collected imaging data and associate them to the donor’s genotypes. We also used in-house tools for cell segmentation and further analysis as well as quality control (Paavolainen group). Primary results based on the features extracted from acquired images showed promising cell type - and donor -type specific clustering.

The TTN gene is composed of 364 exons (363 coding) and encodes for titin, the largest protein in nature. Pathogenic TTN variants result in a wide spectrum of skeletal muscle and cardiac disorders known as Titinopathies. These differ in inheritance patterns, onset age, disease course and severity. The biological mechanisms underlying disease-causing variants specific to titinopathy patients are still elusive. Investigating gene signatures causing the biological pathomechanisms is crucial for understanding genotype-phenotype corelations. RNA-sequencing emerges as a valuable technique for analysing transcriptomic data and exploring gene expression profiles of patient and control samples. To elucidate common pathomechanisms in titinopathies, including adult tibial muscular dystrophy (TMD) due to heterozygous FINmaj variant, and biallelic recessive titinopathies, an extensive differential gene expression (DGE) analysis was conducted using three RNA cohorts from human muscle biopsies. The cohorts involved two polyA enriched and one rRNA depleted batch-corrected cohort. Human DGE analysis identified 265 commonly upregulated genes and 147 commonly downregulated genes in the titinopathy cohorts. A significant downregulation of TTN expression levels was observed in one of the cohorts. To validate and understand the biological significance of these findings, data from a mouse model was incorporated with homozygous Ttn FINmaj variants. Common genes among all cohorts accounted for the structural integrity of the extracellular matrix. This study indicates the pathomechanisms for a skeletal muscle pathology and discusses the future steps in efficiently performing RNA-Seq for titinopathies.

Distal myopathies are a group of rare progressive genetic muscle disorders that are extremely varied both genetically and clinically. Typical symptoms include weakness and atrophy limited to the skeletal muscles of distal extremities in hands and legs. The age of onset ranges from early childhood to late adulthood depending on the disease. Currently around 30 genes have been associated with distal myopathies, most of them causing a dominant disease. The objective of the thesis was to identify the disease-causing variant in a family affected by autosomal dominant distal myopathy with early adulthood onset. Affected family members expressed weakness and atrophy in muscles of both hands and legs. To narrow down the chromosomal location of the disease-causing variant, linkage analysis was conducted with genome-wide single nucleotide polymorphism data of family members. Because of the progressive nature of the disease and uncertain disease status of one family member, linkage analysis had to be repeated a few different times with different settings. Both disease statuses and pedigree size were altered to account for the possibility of presymptomatic carriers or incomplete penetrance. Analyses with different parameters led to discovery of multiple possible co-segregating regions. Rare co-segregating small-scale and structural variants as well as repeat expansions in these regions were examined from next-generation sequencing data with multiple bioinformatic detection tools. The segregation of possible candidate variants was validated with Sanger sequencing and PCR. Ultimately, no likely rare co-segregating variant of any type of genetic variation with a likelihood to cause a disease such as distal myopathy was identified by any detection method used. Lack of potential disease-causing variant could be due to incomplete penetrance of the variant or if it was in non-coding regions, such as a deep intronic splicing variant in a gene currently not known to be connected to muscles.