| 1. |
- Björn, Niclas, et al.
(author)
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Comparison of Variant Calls from Whole Genome and Whole Exome Sequencing Data Using Matched Samples
- 2018
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In: Journal of Next Generation Sequencing & Applications. - 2469-9853. ; 5:1, s. 1-8
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Journal article (peer-reviewed)abstract
- Whole exome sequencing (WES) has been extensively used in genomic research. As sequencing costs decline it is being replaced by whole genome sequencing (WGS) in large-scale genomic studies, but more comparative information on WES and WGS datasets would be valuable. Thus, we have extensively compared variant calls obtained from WGS and WES of matched germline DNA samples from 96 lung cancer patients. WGS provided more homogeneous coverage with higher genotyping quality, and identified more variants, than WES, regardless of exome coverage depth. It also called more reference variants, reflecting its power to call rare variants, and more heterozygous variants that met applied quality criteria, indicating that WGS is less prone to allelic drop outs. However, increasing WES coverage reduced the discrepancy between the WES and WGS results. We believe that as sequencing costs further decline WGS will become the method of choice even for research confined to the exome.
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| 2. |
- Björn, Niclas, 1990-
(author)
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Pharmacogenetic biomarkers for chemotherapy-induced adverse drug reactions
- 2019
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Doctoral thesis (other academic/artistic)abstract
- Cancer is a serious disease expected to be the world-leading cause of death in the 21st century. The use of harsh chemotherapies is motivated and accepted but, unfortunately, is often accompanied by severe toxicity and adverse drug reactions (ADRs). These occur because the classical chemotherapies’ common modes of action effectively kill and/or reduce the growth rate not only of tumour cells, but also of many other rapidly dividing healthy cells in the body. There are also considerable interindividual differences in ADRs, even between patients with similar cancers and disease stage treated with equal doses; some experience severe to life-threatening ADRs after one dose, leading to treatment delays, adjustments, or even discontinuation resulting in suboptimal treatment, while others remain unaffected through all treatment cycles. Being able to predict which patients are at high or low risk of ADRs, and to adjust doses accordingly before treatment, would probably decrease toxicity and patient suffering while also increasing treatment tolerability and effects. In this thesis, we have used next-generation sequencing (NGS) and bioinformatics for the prediction of myelosuppressive ADRs in lung and ovarian cancer patients treated with gemcitabine/carboplatin and paclitaxel/carboplatin.Paper I shows that ABCB1 and CYP2C8 genotypes have small effects inadequate for stratification of paclitaxel/carboplatin toxicity. This supports the transition to whole-exome sequencing (WES) and whole-genome sequencing (WGS). Papers II and IV, respectively, use WES and WGS, and demonstrate that genetic variation in or around genes involved in blood cell regulation and proliferation, or genes differentially expressed at chemotherapy exposure, can be used in polygenic prediction models for stratification of gemcitabine/carboplatininduced myelosuppression. Paper III reassuringly shows that WES and WGS are concordant and mostly yield comparable genotypes across the exome. Paper V proves that single-cell RNA sequencing of hematopoietic stem cells is a feasible method for elucidating differential transcriptional effects induced as a response to in vitro chemotherapy treatment.In conclusion, our results supports the transition to genome-wide approaches using WES, WGS, and RNA sequencing to establish polygenic models that combine effects of multiple pharmacogenetic biomarkers for predicting chemotherapy-induced ADRs. This approach could be applied to improve risk stratification and our understanding of toxicity and ADRs related to other drugs and diseases. We hope that our myelosuppression prediction models can be refined and validated to facilitate personalized treatments, leading to increased patient wellbeing and quality of life.
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| 3. |
- Haage, Pernilla, 1982-, et al.
(author)
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Enantioselective pharmacokinetics of tramadol and its three main metabolites; impact of CYP2D6, CYP2B6, and CYP3A4 genotype
- 2018
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In: Pharmacology Research & Perspectives. - : John Wiley & Sons. - 2052-1707. ; 6:4
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Journal article (peer-reviewed)abstract
- Tramadol is a complex drug, being metabolized by polymorphic enzymes and administered as a racemate with the (+)- and (-)-enantiomers of the parent compound and metabolites showing different pharmacological effects. The study aimed to simultaneously determine the enantiomer concentrations of tramadol, O-desmethyltramadol, N-desmethyltramadol, and N,O-didesmethyltramadol following a single dose, and elucidate if enantioselective pharmacokinetics is associated with the time following drug intake and if interindividual differences may be genetically explained. Nineteen healthy volunteers were orally administered either 50 or 100 mg tramadol, whereupon blood samples were drawn at 17 occasions. Enantiomer concentrations in whole blood were measured by LC-MS/MS and the CYP2D6,CYP2B6 and CYP3A4 genotype were determined, using the xTAG CYP2D6 Kit, pyrosequencing and real-time PCR, respectively. A positive correlation between the (+)/(-)-enantiomer ratio and time following drug administration was shown for all four enantiomer pairs. The largest increase in enantiomer ratio was observed for N-desmethyltramadol in CYP2D6 extensive and intermediate metabolizers, rising from about two to almost seven during 24 hours following drug intake. CYP2D6 poor metabolizers showed metabolic profiles markedly different from the ones of intermediate and extensive metabolizers, with large area under the concentration curves (AUCs) of the N-desmethyltramadol enantiomers and low corresponding values of the O-desmethyltramadol and N,O-didesmethyltramadol enantiomers, especially of the (+)-enantiomers. Homozygosity of CYP2B6 *5 and *6 indicated a reduced enzyme function, although further studies are required to confirm it. In conclusion, the increase in enantiomer ratios over time might possibly be used to distinguish a recent tramadol intake from a past one. It also implies that, even though (+)-O-desmethyltramadol is regarded the enantiomer most potent in causing adverse effects, one should not investigate the (+)/(-)-enantiomer ratio of O-desmethyltramadol in relation to side effects without consideration for the time that has passed since drug intake.
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| 4. |
- Jakobsen, Ingrid, 1984-
(author)
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Prognostic markers in acute myeloid leukemia : A candidate gene approach
- 2018
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Doctoral thesis (other academic/artistic)abstract
- The standard treatment of acute myeloid leukemia (AML) consists of induction chemotherapy, most commonly daunorubicin together with the nucleoside analogue cytarabine (Ara-C), followed by consolidation chemotherapy and in selected cases allogenic stem cell transplantation (allo-SCT). Despite a high initial response rate, a considerable proportion of all AML cases eventually suffer from relapse and the five-year overall survival rate in patients >60 years is only around 15%. Based on cytogenetic analysis, patients are divided into low risk, intermediate risk, and high-risk groups. While low risk patients have a high chance of reaching and remaining in remission after standard induction therapy, high-risk patients are likely to suffer from relapse and should be scheduled for allo-SCT when first complete remission is reached. The intermediate risk group consists of normal karyotype (NK) patients and those with karyotypes of uncertain clinical relevance, but the outcomes are heterogeneous. In NKAML patients, risk classification has improved with the addition of molecular markers including FLT3 internal tandem duplications (ITD) and mutations of NPM1 and CEBPA. Despite this development, there is a group of patients lacking reliable prognostic markers and in some cases the outcomes predicted do not match the outcomes observed, highlighting the need for additional markers. ABCB1 encodes a transporter protein responsible for the extrusion of cytotoxic compounds, including daunorubicin, over the cell membrane, and is a known resistance mechanism. Ara-C is subject to both activating and inactivating metabolic enzymes including DCK (activating), CDA and cN-II (inactivating). ABCB1, DCK, CDA and cN-II are all polymorphic, and SNPs affecting enzyme function and/or activity have potential as prognostic markers. In addition, recurrent IDH1/2 mutations lead to the expression of an enzyme with neomorphic activity associated with epigenetic alterations and disturbed differentiation. Mutations as well as a SNP in codon 105 of IDH1 have prognostic implications in AML, although the effects of different IDH mutations have been unclear. The aim of this thesis was to investigate SNPs in ABCB1 and genes associated with Ara-C metabolism, mutations in IDH1/2 and the IDH1 SNP, and their associations with treatment response and survival in AML. We show that the 1236C>T and 2677G>T SNPs in ABCB1 influence in vitro sensitivity towards AML drugs, with corresponding effects on NK-AML patient survival. These survival differences were seen mainly in patients lacking FLT3-ITD, further adding to the risk stratification. In contrast, the CDA SNPs 79A>C and -451C>T appear to influence survival mainly in FLT3-ITD positive cases. In conclusion, the above-mentioned SNPs have the potential to add important information to risk classifications especially in NK-AML patients with the ambiguous FLT3-ITD-/NPM1- or FLT3-ITD+/NPM1+ genotypes. In addition, we have shown that IDH2 R140 mutation is associated with impaired survival in AML, and that the IDH1 codon 105 SNP appears to confer a worse outcome in a subset of intermediate risk patients without FLT3-ITD. With the introduction of next generation sequencing into clinical diagnostics, IDH mutations may not only provide prognostic information but also guide the selection of patients for new drugs targeting the variant enzyme. Our results indicate that in addition to leukemia-specific mutations, constitutional SNPs may prove useful for further individualizing care-taking and should be considered when implementing these new techniques.
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| 5. |
- Sidstedt, Maja, et al.
(author)
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Assessing the GeneRead SNP panel for analysis of low-template and PCR-inhibitory samples
- 2017
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In: Forensic Science International: Genetics Supplement Series. - : Elsevier BV. - 1875-1768 .- 1875-175X. ; 6, s. 267-269
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Journal article (peer-reviewed)abstract
- Massive parallel sequencing (MPS) is increasingly used for human identification purposes in forensic DNA laboratories. Forensic DNA samples are by nature heterogeneous and of varying quality, both concerning DNA integrity and matrices, creating a need for assays that can handle low amounts of DNA as well as impurities. Commercial short tandem repeat (STR) analysis kits for capillary electrophoresis-based separation have evolved drastically over the past years to handle low-template samples and high amounts of various PCR inhibitors. If MPS is to be used extensively in forensic laboratories there is a need to ascertain a similar performance. We have evaluated the GeneRead Individual Identity SNP panel (Qiagen) that includes 140 SNP markers, following the GeneRead DNAseq Targeted Panels V2 handbook for library preparation, applying low levels of DNA and relevant impurities. Analysis of down to 0.1 ng DNA generated SNP profiles with at least 85% called SNPs, after increasing the number of PCR cycles in the initial PCR from 20 to 24. The SNP assay handled extracts from four different DNA extraction methods, including Chelex with blood and saliva, without detrimental effects. Further, the assay was shown to tolerate relevant amounts of inhibitor solutions from soil, cigarettes, snuff and chewing gum. In conclusion, the performance of the SNP panel was satisfactory for casework-like samples.
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| 6. |
- Watanabe, Shimpei, et al.
(author)
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Biotransformation of the New Synthetic Cannabinoid with an Alkene, MDMB-4en-PINACA, by Human Hepatocytes, Human Liver Microsomes, and Human Urine and Blood
- 2019
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In: AAPS Journal. - : SPRINGER. - 1550-7416. ; 22:1
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Journal article (peer-reviewed)abstract
- Although at a slower rate, new psychoactive substances continue to appear on the illicit drug market, challenging their detection in biological specimens by forensic and clinical toxicologists. Here, we report in vitro and in vivo metabolism of a new synthetic cannabinoid, methyl 3,3-dimethyl-2-[1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido] butanoate (MDMB-4en-PINACA). This is the first report on metabolism of a synthetic cannabinoid with an alkene functional group at the alkyl side chain. MDMB-4en-PINACA was incubated with both human hepatocytes and human liver microsomes (HLM) for up to 5 h and 1 h, respectively. The samples were analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry. An authentic human urine and a corresponding blood sample were analyzed to confirm the in vitro metabolites. A total of 32 metabolites were detected, of which 11 metabolites were detected in hepatocyte samples, 31 in HLM, 2 in urine, and 1 in blood. Analysis of the metabolites revealed that the main metabolic pathway of the terminal alkene group of the pentenyl side chain is dihydrodiol formation, most likely via epoxidation. The majority of the metabolites were generated from ester hydrolysis and/or dihydrodiol formation with further hydroxylation and/or dehydrogenation. Two most abundant metabolites in hepatocyte incubation samples, M8 (ester hydrolysis and dihydrodiol) and M30 (ester hydrolysis), coincided the two detected urinary metabolites. Based on the results, M8 and M30 are proposed to be appropriate urinary markers for MDMB-4en-PINACA intake for screening, while the inclusion of the parent drug itself and M29 (hydroxylation) may be useful for confirmation purposes.
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