Browsing by study line "Genetics and genomics"

Breast cancer is the most prevalent cancer in women worldwide and in 2020 it was the fifth deadliest. In Finland 2019 more than 5000 breast cancer cases were diagnosed, 94% in women and 6% in men. Until now, the high-risk breast cancer susceptibility genes have been identified including BRCA1, BRCA2 and TP53 as well as many of the moderate risk genes. Still, together all the identified genes explain only approximately half of the familial breast cancer cases. Furthermore, all the known breast cancer susceptibility genes are linked to the DNA repair mechanism. Serpina3 stands out as a non-DNA repair gene but as a gene that encodes a protease inhibitor which belongs to the serpin superfamily. Serpina3 has been associated with various diseases before and especially changes in its expression levels are linked to the tumor prognosis in many cancers including breast cancer. However, a previous study proposed that Serpina3 c.918-1G>C is a susceptibility variant for breast cancer in the Northern Finland population. This thesis a case-control study to investigate whether Serpina3 c.918-1G>C variant is associated with breast cancer in the Southern Finland population. In addition, the tumor histology and cellular markers of Serpina3 c.918-1G>C carriers were examined. This study utilized DNA collected from breast cancer patients as well as DNA from blood donors and healthy biobank controls. Breast cancer patients included both familial and unselected cases. The prevalence of Serpina3 c.918- 1G<C variant was studied by genotyping the cases and controls. Genotyping was done by TaqMan real-time PCR and carriers were further confirmed by Sanger sequencing. Moreover, statistical tests were used in the data analyses. The studied Serpina3 c.918-1G>C variant was not found to be significantly (p>0.05) enriched in the breast cancer cases. The variant was found in 0.23 % of familial and 0.36 % of unselected cases, altogether in 0.28 % of all studied breast cancer cases, the frequency in controls was 0.27 %. The tumor histology was found to be ductal in 73 % of the Serpina3 c.918- 1G>C variant carriers and only 9 % had lobular tumor. In other words, the tumor histology followed the usual distribution. All the carriers had a HER2 negative tumor and all except one case were both ER and PR positive. About half of the carriers expressed the cellular proliferation marker Ki67. As a conclusion, the results from this study do not suggest Serpina3 c.918-1G>C as a breast cancer risk variant at least in the Southern Finland population.

The field of gene technology, which falls under the umbrella of biotechnology, presents challenges in business development and commercialisation. Understanding the field characteristics is crucial for successful commercialisation, as it can significantly impact the available strategies for bringing products or services to market, ultimately shaping the business model. This study aims to investigate and understand the challenges associated with commercializing gene technology, including identifying any typical challenge profiles specific to the field and possibly arising from the biological material. The research involves semi-structured interviews with multiple companies, venture capitals, and experts in the field to gain a comprehensive understanding of the challenges. The collected data is then analysed to identify common characteristics and business practices against a commercialisation model frame. The motivation behind this study is to provide researchers and other stakeholders with insights into the challenges they may face while commercializing gene technologies, with the aim of lowering the threshold for business creation. The findings reveal that there are two major groups of companies, each with their specific challenges. The challenges for the major group revolve around business know-how, HR, and sales, while the minor group faces challenges related to technology and regulation. However, a common theme is the limited market awareness among customers, which requires significant efforts in sales, marketing, and communications. The study provides guidance to company founders on the different challenges they should be prepared for and offers insights to society on how to harness the value of gene technologies.

Uterine leiomyomas (ULs) are common benign tumors that originate from the smooth muscle cells of the uterine wall known as the myometrium. Around 70% of pre-menopausal women are affected by these tumors. The high prevalence of ULs is a significant public health issue and ULs are the leading cause for hysterectomy. Many tumors remain asymptomatic, but 15-30% of affected women develop symptoms ranging from pain and heavy menstrual bleeding to pregnancy complications and infertility. Despite their common occurrence, the underlying mechanisms of UL genesis are still largely unknown. Based on mutually exclusive recurring genetic alterations, ULs can be divided into molecular subclasses. Three main molecular subclasses have been established: MED12 mutated tumors, HMGA2 overexpressing tumors and tumors with biallelic FH inactivation. Combined, these three subclasses represent around 90% of ULs, indicating that additional smaller molecular subclasses also exist. Recently, novel mutations associated with ULs have been identified in genes encoding for subunits of the SRCAP chromatin remodeling complex that deposits histone variant H2A.Z onto chromatin. These included loss-of-function mutations in YEATS4, DMAP1 and ZNHIT1, and resulted in deficient H2A.Z loading in the tumors. The detailed functional consequences of these driver mutations need to be further investigated to fully understand their significance in UL genesis. This work aimed to elucidate the effects of YEATS4 mutations by characterizing previously established CRISPR-Cas9 edited immortalized human myometrial cell models carrying heterozygous mutations in YEATS4 using various molecular biology methods. Subcellular fractionation and western blot analysis was used to detect chromatin bound H2A.Z from cell lysates. Quantitative PCR was performed to determine relative YEATS4 expression levels in mutated and wild-type cells. No significant reduction of chromatin bound H2A.Z or YEATS4 expression was observed in the studied heterozygous mutants when compared to wild-type immortalized myometrial smooth muscle cells. Additional myometrial cell models were created by CRISPR-Cas9 gene editing. Objective was to achieve homozygous YEATS4 mutations to better reflect the changes previously reported in ULs. One homozygous YEATS4 mutant cell line was achieved. Understanding the detailed molecular mechanisms behind UL genesis will be instrumental for developing curative non-invasive therapies in the future. Insight into dysregulated pathways and identification of UL biomarkers could improve diagnostic accuracy and help design personalized targeted therapies effective for specific UL subclasses. Characterization of each molecular subclass offers a unique opportunity to understand UL genesis.

Cytokine release syndrome is a severe systematic inflammatory disease that can be triggered upon pharmaceuticals intake. Evaluating the potential risk levels of novel therapeutics with an optimal assay is therefore essential. In this study, we tried to set up and validate a cytokine release assay from human peripheral blood mononuclear cells (PBMCs) for its application in nonclinical immunotoxicity assessments. Fresh PBMCs were isolated from buffy coats obtained from 11 healthy donors of different characteristics. Freshly isolated PBMCs were treated with LPS, positive control antibodies (anti-CD28, anti-CD3) and their corresponding isotypes (negative control antibodies) in both aqueous and solid formats to assess their abilities to induce cytokine release. Similarly, cryopreserved frozen PBMCs were also incubated with LPS, the positive control antibodies and the negative control antibodies, and compared their cytokine releasing capacities with freshly isolated PBMCs. A nine-cytokine panel (IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNFα, IL-12) was screened to select four cytokines (IFNγ, IL-2, IL-6, TNFα) in the following experimental setup. Freshly isolated PBMCs appeared to have higher sensitivity in response to the treatments as shown by the higher level of cytokine release. However, similar trends of cytokine release were observed between freshly isolated and frozen PBMCs in both aqueous and solid assay formats. LPS and anti-CD3 strongly induced cytokine release in all donors. Conversely, anti-CD28 induced cytokine release in some, but not all donors, possibly due to donor specificity. In summary, we have successfully developed and optimized a cytokine release assay, and it can be used to test the potential risk of immune-modulating drug candidate in the preclinical safety studies.

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.

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.

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.

The composition and dynamics of the early life gut microbiota plays a major role in establishing neonatal immunity and is suggested to have multiple impacts on the child’s long-term health. Meanwhile, the composition of the infant gut microbiome has been shown to be affected by the birth mode, infant health and diet. However, the characterization of the infant gut microbiome and its impact on the host’s health is still challenging as the contribution and importance of multiple co-factors on the early microbiome during infant growth is still poorly understood and characterized. The Health and Early-life microbiota (HELMi) is a cohort of more than 1000 healthy Finnish infants currently followed from birth to 4-5 years old. By now, the HELMi dataset comprises more than 400 whole genome shotgun metagenomes obtained from stool samples from 80 infants and parents, but also an in-depth characterization of the families’ lifestyle, environment, health and nutrition, allowing for a precise and cutting-edge characterization of the early gut microbiota. Based on the datasets from the HELMi, this project used Metaphlan3, Kraken and Braken to determine the best computational approach for the taxonomic profiling of the metagenomic reads. Then a PERMANOVA test was performed to evaluate and determine the factors significantly associated with the compositional microbiota variation within the infant gut metagenomes. This study first identified technical factors introducing bias in taxonomic profiling (e.g., DNA extraction batch), which served as confounders in the analysis of environmental and host variables. The investigation of these biological factors indicates that pre-natal and peri-natal variables such as the mode of delivery significantly impact the infant gut microbiota, while we did not identify any significant impact of breastfeeding habits and medication exposures in this study.

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.