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- Grundberg, Ida, 1982-, et al.
(författare)
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Diagnostic mutation testing in situ in routine FFPE tissue sections for treatment prediction in clinical oncology
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Activating mutations in the KRAS gene are present in different cancer types and are strongly associated with resistance to epidermal growth factor receptor (EGFR) inhibitor therapy. Hence there is a requirement for sensitive KRAS mutation analysis to determine the most suitable treatment for the patients. Also, little is known about the impact of tumor heterogeneity with regard to KRAS mutation status in different sub-clones during tumorigenesis, and if this is important for treatment response and prognosis. To improve the diagnostic accuracy, we developed an RNA-based genotyping assay that targets KRAS-mutations in codon 12 and 13 in situ on tissue samples by the use of multiple mutation specific padlock probes and rolling-circle amplification. Thus, the distribution of wild-type (green rolling-circle products) and mutated (red rolling-circle products) KRAS alleles can be determined for single cancer cells in different parts of a heterogeneous tumor without the use of microdissection. We demonstrate reliable detection of KRAS point mutations on cytologic tumor imprints as well as on fresh frozen and formalin-fixed paraffin-embedded tissue sections from colorectal and lung cancer. This in situ method offers single cell mutation detection for diagnostics and holds great promise as a tool to investigate the role of oncogenic mutations in complex tumor tissues.
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- Pottmeier, Philipp, 1987-, et al.
(författare)
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Sex-biased Gene Expression During Neural Differentiation of Human Embryonic Stem Cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Sex differences in the adult human brain are mainly attributed to hormonal influence. Recently however, genetic differences and their impact on the developing nervous system have attracted increasing attention. In order to understand the genetically driven sexual dimorphisms, we investigated genome wide gene expression in an in vitro differentiation model of male and female human embryonic stem cell lines. Four male and four female derived hESC lines were differentiated towards a population of mixed neurons over 37 days. Differential gene expression and gene set enrichment analyses were conducted based on bulk RNA sequencing data. While similar differentiation tendencies in all cell lines proved the robustness and reproducibility of our differentiation protocol, we found sex-biased gene expression already in the undifferentiated ESCs at day 0, but most profound after 37 days of differentiation. Male and female cell lines show sex-biased expression of genes involved in neurodevelopment, suggesting a sex difference in differentiation trajectory. Interestingly, the highest contribution was found to arise from the male transcriptome, involving both Y chromosome and autosomal genes. We propose 13 sex-biased candidate genes (10 upregulated in male cell lines and 3 in female lines) that could strongly affect neuronal development. In addition, we confirmed gene dosage compensation of X/Y homologs escaping X chromosome inactivation through their Y homolog and identified a significantly increased Y homolog overexpression of the gametologs TXLNG/Y and KDM6A/UTY after 37 days of neuronal differentiation. These results suggest sex differences in the trajectories of neuronal differentiation which could ultimately contribute to sex biases during human brain development.
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- Bandaru, Manoj Kumar, 1987-, et al.
(författare)
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Apoc2 mutant zebrafish: a model for hypertriglyceridemia and early-stage atherosclerosis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Zebrafish larvae in a hypertriglyceridemic background can be useful to identify and characterize causal genes for triglyceride metabolism. A previous, small-scale study suggested that apolipoprotein C-II (apoc2)-mutant zebrafish larvae can be used to model hypertriglyceridemia-induced atherosclerosis. We aimed to replicate these findings in a large-scale study and asses if APOC-II may represent a useful therapeutic target. We generated apoc2 mutant zebrafish using CRISPR-Cas9 and examined cardiometabolomic traits in their offspring (F1 generation). Systematic characterization of 384 larvae using our image and assay-based, high-throughput pipeline showed that compound heterozygous larvae for loss of function mutations in apoc2 (n=35) have higher whole-body levels of triglycerides (0.71±0.16 SD), HDL cholesterol (0.32±0.15 SD) and total cholesterol (0.37±0.18 SD), and a trend for lower whole-body glucose levels (0.23±0.14 SD) compared with larvae without mutations in apoc2 (n=174). Such larvae also tended to have more vascular lipid deposition, however this effect did not reach significance (P=0.12). Interestingly, the trends for lower whole-body glucose levels and more vascular lipid deposition in larvae with anticipated loss of functional apoc2 reached significance when larvae (n=3812) from other screens, in which apoc2 was not experimentally perturbed were included as additional wildtype controls. Thus, our large-scale study confirms the role of apoc2 in hypertriglyceridemia and early-stage atherosclerosis. While apoc2 mutant zebrafish model can be used as a genetic background to identify and characterize causal genes for triglyceride metabolism, independent and opposite effects on triglycerides and glucose suggest that APOC-II is likely not a suitable target for prevention and treatment of coronary artery disease.
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- Bandaru, Manoj Kumar, 1987-, et al.
(författare)
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Image-based, in vivo characterization of cardiometabolic consequences of mutations in pcsk9
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Based on the association of loss-of-function mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) with low plasma LDL cholesterol levels, inhibition of the PCSK9 protein using monoclonal antibodies have emerged as an effective treatment option to lower LDL cholesterol levels and reduce the risk of coronary artery disease. Despite these beneficial effects, PCSK9 inhibitors may increase the risk of diabetes. In this study, we mimicked the mechanistic action of PCSK9 inhibitors in humans by inducing mutations in pcsk9 in zebrafish and examining their effects on dyslipidemia, early-stage atherosclerosis and diabetes-related traits in data from nearly 5000 zebrafish larvae. At 10 days of age, larvae with mutations in pcsk9 were characterized by lower whole-body LDL cholesterol levels (beta±SE -0.056±0.025 SD units) and protection against early-stage atherosclerosis, with less vascular lipid deposition (-0.133±0.035 SD) and less co-localization of macrophages with lipids (-0.086±0.032 SD). Mutant larvae also had fewer pancreatic β-cells (-0.153±0.055 SD). Thus, our findings in pcsk9 mutant larvae are in line with results from people carrying loss-of-function PCSK9 mutations, and are also in line with the effects of PCSK9 inhibitors in humans. Further, our results suggest that mutations in pcsk9 may increase the risk of diabetes through a direct effect on pancreatic β-cells.
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- Martinez Barrio, Alvaro, 1977-, et al.
(författare)
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GeneFinder: "in silico" positional cloning of trait genes
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Annan publikation (populärvet., debatt m.m.)abstract
- Motivation: Positional cloning of trait genes is extremely laborious and the amount of information available on gene function in different organisms is increasing so rapidly that it is hard for a research group to collect all the relevant information from a number of data sources without performing a large number of manual and time consuming searches. Results: A web service application named GeneFinder was designed and implemented. It collects selected available information related to trait loci within a given chromosomal region that control a specific phenotype. The information contains details on gene function, disease conditions, tissue expression as well as predicted gene homologies in several other species. The information gathered is further ordered by a special-purpose ranking algorithm. A web interface to the GeneFinder web service was also developed where the results are presented in a ranked list easing its interpretation. We explain the design of the architecture, show how our web interface works, and finally test a candidate region. Availability: GeneFinder is publicly available and free to use. The web interface is available at http://www.genefinder.org/.
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- Johansson, Henrik, 1980-
(författare)
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Multiplex PCR amplification using Extractor probes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Manipulation of a plurality of DNA molecules in parallel offers several advantages over simplex reactions. This paper reports on a new version of Selector probes with easier design procedure and the possibility to achieve higher ROI content within the targeted fragments. To demonstrate its utility we applied it for copy number variance detection and simultaneous amplification of 64 genomic fragments.
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- Badhai, Jitendra, et al.
(författare)
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Differential expression of RPS19 5’UTR variants implicated in Diamond-Blackfan anemia
- 2012
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Heterozygous mutations in the ribosomal protein (RP) S19 gene RPS19 are found in about 25% of patients with the congenital erythroblastopenia Diamond-Blackfan anemia (DBA). The RPS19 gene encodes a single RPS19 isoform from three known transcriptional start sites (TSS) with different 5’ untranslated region (UTR). The regulation of RPS19 expression is poorly understood as well as the significance of different 5’UTRs. A few rare sequence variants within the 5’UTR have also been reported in patients with DBA. We determined the transcriptional start sites (TSS) and the tissue distribution of variant 5’UTRs of RPS19. Twenty-nine novel TSS in K562 cells and testis were identified. We then analyzed the relative proportion of three selected 5’UTRs of different length on a panel of primary tissues. The shorter 5’UTR were most abundant in all tissues but with large variations in relative levels of shorter versus longer transcripts. To clarify the effect of different RPS19 5’UTRs on translation we designed and expressed constructs using three 5’UTRs of different length. The short 5’UTR(+35nt.) translate 4-6 folds more efficiently than the two longer variants with 5’UTRs of 382nt. and 467nt., respectively We also introduced DBA associated insertion (c.-149_-148insGCCA, c.-149_-148insAGCC ) and deletion (c.-144_-141delTTTC) variants in the 5’UTR. . Interestingly, the DBA associated 5’UTR sequence variants showed a 20-30% reduction in RPS19 levels when compared to the corresponding w.t. constructs. Our results indicate that the RPS19 gene has a broad range of TSS with tissue specific variations. We also show that sequence variants in the 5’UTR in some DBA patients reduce RPS19 expression with implications for the pathophysiology of the disease.
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