Browsing by study line "Genetiikka ja genomiikka"

Every year in the western world 3–5% of newborns suffer permanent damages due to prenatal alcohol exposure. Alcohol causes the symptoms of Fetal Alcohol Spectrum Disorders (FASD), which consist of various structural, cognitive, and behavioral neurological defects and distinctive craniofacial features, although in many cases the condition is undiagnosed. The frequency, amount, and timing of alcohol consumption during pregnancy critically influence the symptoms and their severity. Despite the serious consequences and frequent incidence, there is still no clear information on the etiology of FASD symptoms or the timing specific effects of alcohol. However, it has been hypothesized that the early pregnancy is especially susceptible to environmental exposures, such as alcohol, because there is rapid cell proliferation, cell differentiation, and epigenetic reprogramming taking place in the embryo. Gastrulation is a crucial developmental stage in early embryonic development where the three germ layers, endoderm, mesoderm, and ectoderm form and create a foundation for all further development. The aims of this thesis are to study how alcohol affects the gene expression in undifferentiated human embryonic stem cells (hESCs) compared to cells differentiating into the germ layers, and how the gene expression in each of the germ layers is affected. To study the differentiation in gastrulation, hESCs were differentiated in vitro under alcohol exposure to endoderm, mesoderm, and ectoderm with STEMdiff™ Trilineage Differentiation Kit. Gene expression in differentiated germ layers and undifferentiated hESCs was analyzed with 3’mRNA sequencing. The results show that the number of genes with alcohol-induced differential expression is considerably higher in hESCs than in the germ layers indicating that undifferentiated hESCs are more susceptible to alcohol than differentiating cells, which is in agreement with findings from previous studies. In the germ layers, alcohol affected the expression of many genes involved in developmentally important signaling pathways such as FGF, Wnt, and TGF-β. Each of the germ layers have different gene expression profiles and accordingly, they exhibit a unique response to alcohol. Furthermore, the differentially expressed genes reveal intriguing connections to the FASD phenotype, notably, in ectodermal cells alcohol caused differential expression in many genes related to neurodevelopment.

Schizophrenia is a debilitating psychiatric disorder associated with reduced life expectancy. The biological mechanism of schizophrenia is nebulous; however, many findings point to the central nervous system and neurons, where a reduction in dendritic spines has been indicated by previous research. The genetic findings support the involvement of synapses in the pathogenesis of schizophrenia. To study the biological properties stemming from genetics, relevant model systems and efficient methods are needed. Induced pluripotent stem cell (iPSC) technology offers a robust method for modeling the biological processes underlying schizophrenia. Somatic cells, e.g. fibroblasts, can be reprogrammed back to a pluripotent state resembling embryonic stem cells, and further differentiated into any cell type of the body, which might not be otherwise accessible. This allows establishing and characterizing neuronal cultures from patient and control cell lines, potentially revealing biological differences associated to the disease phenotype. The field of schizophrenia research has adopted iPSC technology and multiple studies have been conducted. These include assessments of synaptic density in the produced neuronal cultures, many of which reported decreased density associated with schizophrenia. In this thesis, a modified version of Nehme et al. (2018) protocol was used to differentiate iPSCs into neurons in co-cultures with human iPSC-derived astrocytes. The overarching aim was to construct an immunocytochemistry (ICC) -based assay to measure synaptic density in the produced co-cultures. First, suitable markers for characterization by ICC were tested and selected. The markers were selected to inform about neuronal identity, maturity, and synapses of the differentiated neurons. Next, the culturing conditions were optimized regarding the cell density and coating of the culturing wells. Finally, to estimate the utility of the assay, a pilot study was performed with three cell lines derived from a healthy control and a monozygotic twin pair discordant for schizophrenia. iPSCs from these cell lines were differentiated into neurons in co-cultures with astrocytes, and then characterized with ICC using selected markers and image analysis software. The synaptic density was quantified for each cell line. The performance of the assay was evaluated with analysis of variance (ANOVA) and restricted maximum likelihood model (RELM). An assay to quantify synaptic structures in mature neurons was established. The average synaptic density for all cell lines was approximately 1 synapse per 100μm of neurite. Analysis of the data produced with the assay revealed a notable batch effect and technical variation. This suggests that further optimization is needed to reduce variance from undesired sources. The pilot data suggests that the differences in synaptic density between cases and controls may be modest, further highlighting the need for minimizing noise in the assay to improve signal to noise ratio. However, indicated by power analysis, large sample sizes are needed to identify meaningful differences between cases and controls. In light of these results, more attention should be drawn to the methodology in the field of iPSC-based studies, as the principals of the assay constructed here were similar to other synaptic assays used in previous publications.

Leaf senescence is a developmental and physiological phase in plants to end leaf development. Environment factors such as drought stress, extreme temperature, and pathogen threat and internal factors including age and reactive oxygen species induce leaf senescence. Some phytohormones such as jasmonic acid and salicylic acid play a key function in cell death in plants. WRKY transcription factors is known as one of the largest transcription factor family in plants which regulates a variety of plants processes. WRKY75 which belong to WRKY transcription factors has shown multiple functions in plant development like regulation of Pi starvation responses and root development and flowering. In my thesis, I focused on the role of WRKY75 in senescence and stress responses. WRKY75 was identified as a positive regulator of cell death in Arabidopsis. WRKY75 can promote salicylic acid biosynthesis by promote transcript levels of SID2 and also cause hydrogen peroxide accumulation by suppressing the transcription of CAT2. Hydrogen peroxide and salicylic acid can promote WRKY75 transcription at the same time. To evaluate the function of WRKY75 transcription factor in SA signalling and cell death, three lesion mimic mutants acd5, cat2, dnd1 and their corresponding wrky75 double mutant were used. Interestingly, no different phenotypes were found between acd5, cat2, dnd1 and their corresponding wrky75 double mutants in cell death and hydrogen peroxide accumulation detection in Arabidopsis leaves. Meanwhile, marker genes transcription levels were not different in both short day and long day growth condition. However, different phenotypes were observed in botrytis infection. Based on these results, we formed a hypothesis that gene redundancy could influence genetic characterization of WRKY75. To overcome this problem, SRDX-WRKY75 chimeric repressor transgenic lines were generated. The SRDX domain act as a dominant negative regulator to suppress WRKY75 target genes. In future research, these new lines can be used to test transcript levels for putative WRKY75 target genes.

Progressive retinal atrophy or PRA is a collective term for a group of hereditary degenerative retinal diseases in dogs. PRA affects the photoreceptor cells of the eye ultimately progressing into complete vision loss. Documented in over 100 breeds, it is the most common type of canine retinal diseases. PRA is considered a homologous disease to human retinitis pigmentosa, thus providing a large animal model for studying retinal biology and genetic aetiology of its diseases. The objective of this thesis was to study the genetic cause of a novel form of PRA in young Finnish Lapphunds. Analysis built upon a combination of gene mapping methods and analysis of next­ generation sequencing data. Gene mapping was performed with two analysis methods, genome­-wide association study and homozygosity mapping, utilising single nucleotide polymorphism microarray based genotype data. Identifying a clinical phenotype from the canine biobank at the University of Helsinki resulted in a study cohort of six case and 10 control dogs. Combined with pedigree information, this early­-onset PRA was most likely a new autosomal recessive condition in the breed. Genome­-wide analyses resulted in the discovery of a disease­-associated locus on chromosome 27. Findings of single nucleotide variant filtering of one whole-­genome sequenced affected dog led to the prioritisation of an intronic substitution variant (T > C) in SOX5 gene as a potential cause of PRA. Genetic validation of the variant with 23 dogs showed promising results. Four out of five affected dogs were homozygous for the variant, while controls were either wild-type or heterozygotes. As a result, a previously unknown disease locus was successfully identified, suggesting a possible new spontaneous canine model of retinitis pigmentosa. By better understanding the patho­physiological processes of disease, improved diagnostics and marker­-based testing as well as novel therapies can be developed for both dog and man. However, further studies are needed to understand the underlying molecular mechanism of the candidate disease variant.

Henoch-Schölein purpura (HSP) is a vasculitis of small vessels and its characteristics include abnormal accumulation of IgA immunocomplexes on vessel walls as well as abnormal glycosylation patterns of IgA. HSP is an autoimmune disease like inflammatory bowel diseases (IBD). The genetic background of HSP has not been studied in Finnish population before, and only one genome-wide association study has been conducted for HSP before. Therefore investigating the Finnish genetic associations of HSP on a genome-wide level is of value. In this study the genetic background of HSP is studied with genome-wide association analyses performed on 424,041 genotyped SNPs passing quality control, HLA alleles imputed from the SNPs, and for their allele-level HLA protein sequences with the aim of replicating previous HSP associations in a Finnish cohort. There were 46 HSP individuals and 18,757 controls (216 bone marrow donors and 18,541 blood donors) passing quality control and included in the study. R package HIBAG was used for HLA imputation, and SPAtest package was used for the association analyses. In the association analyses, a region in chromosome 6 passed genome-wide significance (SNP with the smallest p-value: p 6,57 x 10-10, OR 0.14[0.1-0.2]) and the region contained both predisposing and protective associations. Of HLA alleles, DQB1*05:01, DQA1*01:01 ja DRB1*01:01 surpassed genome-wide significance level (p values 4,99 x 10-9, 1,04 x 10-8 and 2,37 x 10-8, respectively) and were positively associated with HSP. Five amino acid positions were significantly associated with HSP (p-values 3,9 x 10-10, 7,37 x 10-9, 1,26 x 10-8, 1,69 x 10-8 and 2,41 x 10-8), being both protective and predisposing to HSP. In addition, the genetic background of HSP was compared with that of IBD by comparing their GWAS results of genotyped SNPs, HLA alleles and their protein sequences. There were 49 IBD patients after quality control, and the same controls as for HSP (18,541 individuals) were included in the association analyses of IBD. The diseases seem to share some of their genetic background. According to the results, HSP seems to associate primarily with HLA class 2 and the result is also compatible with previous studies linking HSP to this region. The results also replicate previous GWAS findings in HLA class 2. According to this it is likely that the same HLA alleles are notable genetic factors in both Finnish and Spanish populations. The connection between HSP and IBD could potentially have to do with intestinal microbes aiding the onset of autoimmune diseases in genetically susceptible hosts.

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.

Puberty initiation is a crucial physiological process in human development. A group of hypothalamic neurons secreting the gonadotropin-releasing hormone (GnRH) and expressing the kisspeptin receptor (KISS1R) plays a key role in launching puberty. Furthermore, cellular KISS1R signaling has been shown to regulate GnRH expression and secretion. Although the in vitro differentiation of human pluripotent stem cells into GnRH-secreting neurons has been successful, it is of high interest to generate KISS1R expressing GnRH neurons. By utilizing the CRISPR activation technology, this study aimed to establish a conditional KISS1R-activation cell line using H9 human embryonic stem cells. Through controlling dCas9VP192 abundance using the Tet-On system combined with the dihydrofolate reductase destabilizing domain, the transcriptional activation of KISS1R was temporally regulated by the addition of two antibiotic drugs - doxycycline and trimethoprim. KISS1R expression was primarily assessed by qPCR and verified by immunocytochemistry and the use of a KISS1R-GFP reporter cell line. The main finding of this study is the achievement of a 6217 ± 2286 fold change in KISS1R transcription by introducing two guide RNAs (N = 3). Nevertheless, leaky gene activation was observed without drug treatment (fold change of 63 ± 51). Concludingly, this study successfully led to the generation of a KISS1R-activation cell line. After further characterization and refinement of the activation protocol, the established cell line will enable to investigate whether KISS1R upregulation modulates in vitro GnRH neuron differentiation, electrophysiology, hormone expression, and secretion in the future. Respective outcomes may lead to advances in understanding and treating pubertal disorders.

Elevated low-density lipoprotein cholesterol (LDL-C), hypercholesterolemia, is characterized by complex and poorly understood genetic contributions. Cellular LDL uptake mediated by the LDL receptor is pivotal to disease progression. After LDL internalization LDLR is recycled to the plasma membrane. Genetic mutations are known to exist in factors driving LDLR recycling but their contribution to hypercholesterolemia is not known. SNX17 has been postulated to be important for LDLR recycling. The goal of this study was to investigate the effect of SNX17 on cellular LDL uptake and to evaluate whether functional characterization of SNX17 gene variants can be performed. At the same time, adjusting an existing semi-automated analysis pipeline to generate expression constructs for SNX17 genetic variants. In this study, using an SNX17 knock-out cell line and an SNX17 rescue cell line (SNX17 knock-out cells transfected with GFP-SNX17 construct), it was shown that SNX17 might have a role in LDL uptake. The semi-automated workflow for generating genetic variants was successfully adopted to SNX17, warranting further experiments to define the optimal conditions for the functional characterization of SNX17 gene variants. This thesis sets the foundation for a deeper understanding of SNX17 in LDLR recycling and provides first insights into the potential regulation of this pathway, while also initiating the way for the later characterization of SNX17 variants. Hence, functional genomic studies together with the functional characterization of genetic variants in LDLR recycling factors can improve our understanding of how genetic variation contributes to disease progression and develop better risk assessment tools.

Long interspersed nuclear element 1 (LINE-1 or L1) belongs to a class of retrotransposons. In other words, it is a DNA element that can copy and paste itself around the genome. There are approximately 500,000 copies present in humans, but only around 5,000 are expected to remain transcriptionally competent. The activity of L1s is generally strongly repressed in normal human tissues, but in many cancers, these elements are reactivated. Both L1 transposition and transcription can have significant effects on cellular function, making it an interesting topic of research from a pathological point-of-view. By studying and understanding more about this transposon, it could be possible to find novel screening methods or even therapeutics for different cancers. One of these cancer types is high-grade serous ovarian carcinoma (HGSOG), which is known for exhibiting L1 upregulation. However, the quantification of L1 transcription has been proven to be very challenging, mostly due to alignment issues caused by the repetitive nature of the element. In addition, a large proportion of L1s reside within genes, meaning that L1 sequence -containing transcripts frequently do not originate from the L1’s own promoter. This thesis aimed to tackle these challenges; I quantified L1 expression at the single-locus level in 11 pre- and post-chemo HGSOC sample pairs, as well as in 5 samples from healthy women, based on single-cell RNA-sequencing. In addition to comparing L1 activity in different sample and cell types, I researched whether L1 activity was associated with any changes in gene expression. The poly(A) site of an L1 is relatively weak, meaning that L1 transcription frequently extends over it. Based on this fact, the utilized approach was to quantify L1 expression based on reads mapping to the 1 kilobase downstream window of each L1 locus, thus minimizing the alignment issues of repetitive elements. Thereafter, the features of the detected loci were carefully assessed to separate false-positive L1s from those with evidence supporting genuine activity, such as tumor sample enriched expression, lack of correlation to host gene, and detection with bulk RNA-sequencing. The activity of the latter loci was then further analyzed to search for differences in L1 expression between pre- and post-chemo samples. In addition, the association between L1-activity and gene expression was examined based on regression models both at the individual gene and molecular signature gene set-level. It was found that L1 expression data is filled with factitiously active loci, highlighting the importance of careful analysis and wet lab validations when studying transposon activity. However, regardless of the issues arising from a sparse and unreliable dataset, I showed that L1 activity was negatively associated with the expression of MYC target genes. MYC has been previously shown to be a transcriptional repressor of the L1, indicating that the obtained results are legitimate. Even though the results obtained from this study appear to be biologically justifiable, they would require further validation to ensure their authenticity. In addition, for the future it would be essential to enhance the sensitivity of the utilized workflow to minimize the sparsity of the data, so that statistical analyses performed would become more reliable. Nevertheless, it was shown that assessing L1 expression at the single-cell level using RNA-sequencing is executable.

Neurodevelopmental disorders (NDDs) are disabilities in which the formation and development of the central nervous system is altered. NDDs severely impact the quality of life of the individuals that are affected by them, however little is known about the causes or the molecular mechanisms that are behind their onset. For this reason, being able to model them is pivotal to our society since, by understanding the mechanisms underlying such disorders, we could develop possible treatments. Previous research has suggested that disturbances in the early neuronal development could be at the basis of NDDs onset. Therefore, in this work, I have modeled neuronal differentiation in Kabuki syndrome (KS), a known NDD, assaying the expression of key early neurodevelopmental markers at four specific timepoints, using induced pluripotent stem cell (iPSC) technology. By concurrently differentiating three KS patient-derived and three control iPSC lines to neural precursor cells (NPCs) and profiling them with immunocytochemistry (ICC) and quantitative real-time PCR (RT-qPCR), I was able to identify differences in the early developmental trajectories of NPCs between the two conditions. The ICC data suggested that differentiating KS cell lines incur in precocious differentiation when compared to control cell lines, suggesting that the disease-causing mutations could lead to accelerated neuronal maturation of early NPCs. However, RT-qPCR analysis of the expression patterns of key neurogenesis markers was unable to statistically confirm the observed trend between the two phenotypes, likely due to limitations in statistical power. Despite this, the expression of four out of seven NPC markers was higher in early KS cells than in control cell lines, supporting the hypothesis of accelerated neuronal maturation. Taken together, this work highlighted some of the challenges related to iPSC-based disease modelling studies, and the need to further confirm the inferred mechanisms of asynchronous neuronal development observed in this work.