| 1. |
- Etemadikhah, Mitra
(author)
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Gene and pathway associations in neurodevelopmental disorders
- 2021
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Doctoral thesis (other academic/artistic)abstract
- Neurodevelopmental disorders are complex and heterogenous disorders affecting brain development. In this thesis, we study intellectual disability (ID) and schizophrenia, and we applied genomic, transcriptomic, and proteomic techniques to identify and further investigate candidate variants and important genes and pathways involved in pathology of these two disorders.In paper I, genotyping and exome analyses were performed in a large multi-generational family with non-syndromic ID from a North Swedish isolate, with the aim to identify linkage regions and disease-associated variants. The linkage analysis identified four suggestive linkage regions. The exome sequencing revealed a known pathogenic mutation in the SLC17A5 gene, and potential pathogenic CNVs overlapping the genes KDM3B and MAP3K4/AGPAT4. The overall results indicated that ID is genetically heterogeneous in this family. In paper II, transcriptome sequencing was performed on fibroblast cells from schizophrenia patients and control individuals from a large family in a genetically isolated region in northern Sweden. We detected 48 significant differentially expressed genes, of which eight genes were previously associated with schizophrenia. These results provide further support for the use of fibroblasts, and highlight benefit of using isolated populations in studies of neurodevelopmental disorders.In paper III, transcriptome sequencing was performed on a large cohort of post-mortem brain tissue samples from schizophrenia patients and controls. In total, 71 significant differentially expressed genes were detected, and gene ontology analysis showed enrichment of genes from the immune system and more specifically the complement system. Our results implicate significant upregulation of complement genes in a subset of the patients.In paper IV, we performed further proteomic analysis on the same sample set as paper III. The primary results from proximity extension assays show significant differential expression of schizophrenia-associated proteins in a subgroup of patients. In total, 21 differentially expressed proteins were identified by the immune panel, 12 proteins by the inflammation panel, and two by the custom panel. The protein IL6 was included in all three panels and consistently showed significant expression changes. Our transcriptome and proteome results highlight the important role of immune system in schizophrenia pathology.
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| 2. |
- Feuk, Lars
(author)
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SNP based strategies to study candidate genes for Alzheimer’s disease
- 2002
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Doctoral thesis (other academic/artistic)abstract
- Alzheimer’s disease (AD) is the most common form of dementia in the elderly. It is a genetically heterogeneous disease characterized by progressive cognitive decline and memory impairment. The rare familial form of AD is caused by three different genes called APP, PSEN1 and PSEN2. However, the predominant form of AD is a genetically complex disorder involving a combination of genetic factors. To date, the only risk factor identified for the complex form of AD is the APOE-epsilon 4 allele, but several susceptibility genes remain to be found. This thesis outlines different strategies to use common genetic variation, in the form of single nucleotide polymorphisms (SNPs), to examine candidate genes and candidate regions for AD. Large-scale genotyping is a prerequisite for performing complex disease studies using SNPs. The validity and accuracy of a newly developed genotyping assay called Dynamic allele specific hybridization (DASH) was therefore investigated. DASH was shown to be a robust genotyping method, and was proven to work as well or better than several other available methods. 'the method was first implemented for a candidate gene association study of a promoter polymorphism in the TNFRSF6 gene. Significant association was found between this variant and early onset AD, indicating its possible role in disease etiology. A large candidate pathway association study effort was then started testing for association between AD and 60 different SNPs. Genes were picked from four different pathways related to AD; oxidation, inflammation/apoptosis, amyloid interacting genes and a group of candidate genes previously showing significant association with AD. None of the markers showed significant disease association after correction for multiple testing. Although largely negative, these results high-lighted several methodological and study design issues related to association studies in general. The most successful approach yet in dissecting complex disease using genetic variation has been to perform high resolution linkage disequilibrium (LD) mapping of regions indicated by linkage. Several independent research groups recently reported linkage peaks for AD on chromosome 10q We choose two regions under the 10q linkage peak for LD mapping studies. The first region contained the previously associated TNFRSF6 gene, and the other region included the insulin-degrading enzyme (IDE) gene, which has been shown to be involved in clearance of amyloid-beta. LD maps were created for all pair-wise markers in the two regions to determine the genetic LD structure. Haplotypes were estimated and haplotype tagging markers were chosen for further analysis. Association analyses were performed for both single markers and haplotypes for case/control status as well as for quantitative traits related to the AD phenotype. Only weak significant signals were found for the TNFRSF6 gene. However, several significant associations were found for a large LD block including the IDE gene. The same haplotypes were always over-represented in cases compared to controls, or with more severe AD within the patient groups. These results indicate a role in AD for one of the three genes situated within the 276kb LD block including the IDE, KNSLI and HHEX genes. Further studies will now be required to identify the underlying risk alleles within the region.
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| 3. |
- Halvardson, Jonatan, 1982-
(author)
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Sequence based analysis of neurodevelopmental disorders
- 2016
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Doctoral thesis (other academic/artistic)abstract
- In this thesis the main focus is the use of methods and applications of next generation sequencing in order to study three of the most common neurodevelopmental disorders: intellectual disability, epilepsy and schizophrenia. A large fraction of the genes in our genome produce several distinct transcript isoforms through the process of splicing and there is an increasing amount of evidence pinpointing mutations affecting splicing as a mechanism of disease. In Paper I we used exome capture of RNA in combination with sequencing in order to enrich for coding sequences. We show that this approach enables us to detect lowly expressed transcript and splice events that would have been missed in regular RNA sequencing using the same coverage. In Paper II we selectively depleted the different transcripts of Quaking (QKI), a gene previously associated to schizophrenia. Using RNA sequencing we show that the effects of depletion differ between transcripts and that the QKI gene is a potential regulator of the Glial Fibrillary Acidic Protein (GFAP), a gene implicated in several diseases in the central nervous system.De-novo mutations are frequently reported to be causative in neurodevelopmental disorders with a strong genetic component, such as epilepsy and intellectual disability. In Paper III we used exome sequencing in family trios where the child was diagnosed with both intellectual disability and epilepsy, focusing on finding de-novo mutations. We identified several previously unknown disease causing mutations in genes previously known to cause disease and used previously published interaction and mutation data to prioritize novel candidate genes. The most interesting result from this study are the implication of the HECW2 gene as a candidate gene in intellectual disability and epilepsy. In Paper IV we used RNA sequencing of post mortem brain tissue in a large cohort of schizophrenics and controls. In this study we could show that the immune system and more specifically the complement system was dysregulated in a large fraction of patients. Further, using co-expression network we also found some evidence suggesting genes involved in axon development and maintenance.
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| 4. |
- Höijer, Ida
(author)
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Targeted Long-read Sequencing : Development and Applications in Medical Genetics
- 2021
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Doctoral thesis (other academic/artistic)abstract
- Targeted sequencing has the advantage of providing pinpointed DNA information, while costs and data-analysis efforts are reduced. If targeted sequencing is combined with single molecule long-read sequencing, it can become a powerful tool to investigate genomic regions traditionally difficult using the predominantly used short-read sequencing platforms, including repetitive regions and large structural variants.The aim of this thesis has been to develop and apply novel targeted long-read sequencing protocols to solve research questions of biomedical and clinical interest. In Paper I we utilized a new amplification-free targeted long-read sequencing method to study trinucleotide repeats in the huntingtin (HTT) gene, associated with Huntington’s disease. This method generated reads spanning the entire repeats, and we could accurately determine the repeat sizes in patient samples. Moreover, we could discover somatic variation of HTT repeat elements as a result of sequencing single, unamplified DNA molecules. In Paper II we present the Xdrop technology, a microfluidic-based system for targeted enrichment of large DNA molecules in droplets from low input samples. We applied the Xdrop technology to detect human papilloma virus 18 (HPV18) integration sites in the human genome of a cervical cancer cell line by targeting the virus genome. We also demonstrated its utility in detecting and phasing SNVs in the tumor suppressor gene TP53 in leukemia cells. In Paper III we employed targeted long-read sequencing to identify CRISPR-Cas9 off-target mutations in vitro with our two novel methods Nano-OTS and SMRT-OTS. Importantly, we were able to identify Cas9 cleavage sites in regions of the human genome that are difficult or impossible to assess using short-read sequencing. The aim of Paper IV was to investigate large structural variants (SVs) induced by CRISPR-Cas9 at on-target and off-target sites in genome edited zebrafish and their offspring. Nano-OTS was used to identify Cas9 off-target sites for four guide RNAs, which were also used for genome editing of fertilized fish eggs. Aided by long-read re-sequencing, we showed that Cas9 can induce large SVs at both on-target and off-target sites in vivo, and that these adverse variants can be passed on to the next generation.This thesis has highlighted a diversity of targeted long-read sequencing methods and some of their applications in medical genetics. We believe these methods could have an important place in future research and clinical diagnostics, and that the scope of their utility will be far beyond the applications demonstrated in this work.
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| 5. |
- Höjer, Pontus
(author)
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Exploring human variations by droplet barcoding
- 2024
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Doctoral thesis (other academic/artistic)abstract
- Biological variations are being explored at ever-increasing rates through the rapid advancement of analytical techniques. Techniques like massively parallel sequencing empower scientists to accurately differentiate individuals’ genetic compositions, cellular functionalities, and healthy tissue from diseased. The knowledge gained from these techniques brings us ever closer to grasping the complexities of life, contributing to human development. Still, to fully elucidate biological variations in different samples requires novel sensitive and high- throughput techniques, capable of placing everything in its correct context. One such technique gaining promise is droplet barcoding. Droplet barcoding leverages emulsion droplets to segregate samples into their functional components, coupled with barcodes that can group tagged molecules following sequencing. This technique constitutes a versatile tool for studying biological variations in both the phenotype and genotype. This thesis leverages droplet barcoding to explore variations relating to human biology. Droplet barcoding was used to study phenotype variations, looking at protein compositions in single extracellular vesicles (Paper I) and single cells (Paper II). Paper I studies extracellular vesicles which are naturally released from cells. They carry heterogeneous protein signatures that can inform about their cellular origin. Tens of thousands of extracellular vesicles were profiled, including approximately 25,000 from lung cancer patients. From these protein profiles, extracellular vesicles could be grouped into putative subtypes. Paper II presents a novel method for studying single cells which was used to characterize blood-derived immune cells. The method enabled the identification of most major immune cell lineages. Haplotype-resolved genetic variations were analyzed using a linked read sequencing method based on droplet barcoding. Linked-read sequencing conserves long-range information from short-read sequencing by co- barcoding subsections of long DNA fragments. Paper III presents an open-source pipeline (BLR) for whole genome haplotyping using linked reads. BLR generates accurate and continuous haplotypes, outperforming PacBio HiFi-based diploid assembly. We further show that integration with low-coverage long-read data can improve phasing accuracy in tandem repeats. With 10X Genomics linked reads, BLR generated more continuous haplotypes compared to other workflows. Paper IV applies linked read sequencing to reveal the haplotype complexities of cancer genomes. In two patients with colorectal cancer, we identified several large-scale aberrations impacting cancer-related genes. Additionally, several short somatic variants were found to impact nearly all oncogenic networks identified by TCGA. Demonstrating the importance of haplotype-resolved analysis for cancer genomics, one patient exhibited two nonsense mutations on separate haplotypes in the well-known colorectal cancer gene APC.
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| 6. |
- Kvastad, Linda
(author)
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The Spatial Context – through the lens of method development
- 2021
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Doctoral thesis (other academic/artistic)abstract
- In the present moment of time, we find ourselves in a period where the advancement of genomic tools is progressing at a fast pace. Of particular interest for this thesis is the study of gene activity. What patterns of genes are expressed? Where are they expressed? How can we use this knowledge to improve our quality of life? The research presented in this thesis focuses on developing and applying new tools for interrogating cells and tissues. In Paper I, we describe a protocol for transcript profiling of single cells, capable of measuring the relative expression levels for genes of interest. We successfully applied our method to cancer cells from metastatic breast cancer patients. Profiling 2 to 4 single cells per patient and measuring gene-specific expression from targets previously associated with metastatic breast cancer supports the use of our protocol as a diagnostic tool. In Paper II, we present an assay for spatial RNA quality evaluation, used to estimate the success for tissue specimens before proceeding with more expensive spatial sequencing methods. We showed that the method is capable of measuring high RNA quality in tissue areas of both high and low cell density and that the spatial RNA integrity patterns are reflected in spatial transcriptomics data. In Paper III, we present a protocol for performing spatial mRNA genome-wide expression profiling of FFPE tissue specimens. Thus, we bridge a gap between traditional tissue preservation methods and novel gene technologytools. We found a high Pearson correlation of 0.95 between formalin-fixation paraffin embedding (FFPE) and Fresh Frozen (FF) mouse brain datasets. Although the FPPE samples yielded fewer transcripts and genes compared to FF, there was a high agreement in gene expression between paired anatomical areas for FFPE and FF samples. In Paper IV, we present an approach to investigate in situ transcript derivedinferred copy number variation (iCNV) profiles based on spatial transcriptomics data. In a normal lymph node that displays both distinct gene expression patterns and histological landmarks, we observed a neutral iCNV profile. In contrast, we found huge variabilities investigating several malign tissue types ranging from homogenous (pediatric medulloblastoma) to highly variable genomes (ductal breast cancer and glioblastoma). Strikingly, we also observed similar iCNV profiles in both tumor and benign tissue areas from prostate and skin cancer. In Paper V, we explore the transcriptional and genomic landscape in pediatric tumors from 14 patients. Microglia cells have been implicated to play an important role in the tumor microenvironment, and we found spatial co-localization of microglia and epithelial-to-mesenchymal transition (EMT) signatures in our patient cohort. Furthermore, we found homogenous and recurrent iCNV profiles in the high-grade tumors of relapse patients and identified expression of gene SPP1 in the tumor stroma as a potential prognostic mRNA marker in pediatric brain tumor relapse patients.
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| 7. |
- Zaghlool, Ammar, 1980-
(author)
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Genome-wide Characterization of RNA Expression and Processing
- 2013
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Doctoral thesis (other academic/artistic)abstract
- The production of fully mature protein-coding transcripts is an intricate process that involves numerous regulation steps. The complexity of these steps provides the means for multilayered control of gene expression. Comprehensive understanding of gene expression regulation is essential for interpreting the role of gene expression programs in tissue specificity, development and disease. In this thesis, we aim to provide a better global view of the human transcriptome, focusing on its content, synthesis, processing and regulation using next-generation sequencing as a read-out.In Paper I, we show that sequencing of total RNA provides unique insights into RNA processing. Our results revealed that co-transcriptional splicing is a widespread mechanism in human and chimpanzee brain tissues. We also found a correlation between slowly removed introns and alternative splicing. In Paper II, we explore the benefits of exome capture approaches in combination with RNA-sequencing to detect transcripts expressed at low-levels. Based on our results, we demonstrate that this approach increases the sensitivity for detecting low level transcripts and leads to the identification of novel exons and splice isoforms. In Paper III, we highlight the advantages of performing RNA-sequencing on separate cytoplasmic and nuclear RNA fractions. In comparison with conventional poly(A) RNA, cytoplasmic RNA contained a significantly higher fraction of exonic sequence, providing increased sensitivity for splice junction detection and for improved de novo assembly. Conversely, the nuclear fraction showed an enrichment of unprocessed RNA compared to when sequencing total RNA, making it suitable for analysis of RNA processing dynamics. In Paper IV, we used exome sequencing to sequence the DNA of a patient with unexplained intellectual disability and identified a de novo mutation in BAZ1A, which encodes the chromatin-remodeling factor ACF1. Functional studies indicated that the mutation influences the expression of genes involved in extracellular matrix organization, synaptic function and vitamin D3 metabolism. The differential expression of CYP24A, SYNGAP1 and COL1A2 correlated with the patient’s clinical diagnosis.The findings presented in this thesis contribute towards an improved understanding of the human transcriptome in health and disease, and highlight the advantages of developing novel methods to obtain global and comprehensive views of the transcriptome.
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| 8. |
- Zhao, Jin, 1983-
(author)
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Sequence based identification of genetic variation associated with intellectual disability
- 2017
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Doctoral thesis (other academic/artistic)abstract
- Intellectual disability (ID) is a common neurodevelopmental condition, often caused by genetic defects. De novo variation (DNV) is an important cause of ID, especially in severe or syndromic forms of the disorder. Next generation sequencing has been a successful application for finding pathogenic variation in ID patients. The main focus of this thesis is to use whole exome sequencing (WES) and whole genome sequencing (WGS) to identify pathogenic variants in undiagnosed ID patients. In Paper I, WES was used in family trios to identify pathogenic DNVs in patients diagnosed with ID in combination with epilepsy. This work led to the identification of several DNVs in both new and known disease genes, including the first report of variation in the HECW2 gene in association with neurodevelopmental disorder and epilepsy. Paper II is the first independent validation of PIGG as a disease-causing gene in patients with developmental disorder. We used WES to identify the homozygous variation in PIGG, and transcriptome analysis as well as flow-cytometry studies were used to validate the pathogenicity of the PIGG variation. We discovered that PIGG variation give different effects in different cell types, contributing new insights into the disease mechanism. Paper III is also an application of WES in trio families with patients diagnosed with ID in order to identify causal variants, a strategy similar to that of Paper I. Several pathogenic variants were identified in this study; in particular, the gene NAA15 is highlighted as a new disease gene, and was recently confirmed in independent studies. This study also adds evidence to support that variation in the PUF60 gene is causing the symptoms in patients with Verheij syndrome. In Paper IV, WGS was used to analyze families with consanguineous marriages. All families in this study had been previously analyzed with WES without finding a disease cause. A number of new disease-causing variants were identified in the study, including a first validation of FRMD4A as a disease-associated gene. This study also shows that WGS performs better than WES in finding variants, even for variants in coding parts of the genome.
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