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Context: Opioids are the mainstay therapy for patients affected by cancer pain. However, about 10%-20% of patients do not benefit from the received analgesic treatment or experience side effects. Genetic variability might account for the variation in individual responses to opioids, both in terms of efficacy and toxicity., Objectives: The aim of this genome-wide association study (GWAS) was to identify genetic markers of opioid toxicity, in terms of nausea-vomiting., Methods: Cancer patients receiving morphine, oxycodone, buprenorphine, and fentanyl were recruited from different European countries. Data about toxicity (nausea-vomiting score, NVS) and other relevant clinical information were collected, as well as genotyping data. Regression analysis between genotypes of 2052 patients and NVS was performed, using appropriate covariates, with REGENIE software., Results: We found 65 variants associated with NVS (P-value < 1.0×10 -5 ). Of note, 14 intronic variants on chromosome 2 were in NPAS2 gene, encoding a circadian transcription factor reported to play a role in another opioid side effect, the alteration of sleep. Some of these variants were previously identified as splicing quantitative trait loci of the NPAS2 gene., Conclusions: This is the first GWAS, performed in more than two thousand individually genotyped patients treated with opioids for cancer pain, that investigated the genetic bases of opioid-induced nausea-vomiting. Although further studies are needed to confirm our findings and to characterize the functional role of the identified variants, our results emphasize the importance of performing large pharmacogenomic studies to identify germline variants associated with opioid response, with the ultimate goal of tailoring cancer pain therapies., (Copyright © 2024 American Academy of Hospice and Palliative Medicine. Published by Elsevier Inc. All rights reserved.)

Studies have indicated that detection of circulating tumor DNA (ctDNA) prior to treatment is a negative prognostic marker in non-small cell lung cancer (NSCLC). ctDNA is currently identified by detection of tumor mutations. Commercial next-generation sequencing (NGS) assays for mutation analysis of ctDNA for routine practice usually include small gene panels and are not suitable for general mutation analysis. In this study, we investigated whether mutation analysis of cfDNA could be performed using a commercially available comprehensive NGS gene panel and bioinformatics workflow. Tumor DNA, plasma DNA and peripheral blood leukocyte DNA from 30 NSCLC patients were sequenced. In two patients (7%), tumor mutations in cfDNA were immediately called by the bioinformatic workflow. In 13 patients (43%), tumor mutations were not called, but were present in ctDNA and were identified based on the known tumor mutation profile. In the remaining 15 patients (50%), no concordant mutations were detected. In conclusion, we were able to identify tumor mutations in ctDNA from 57% of NSCLC patients using a comprehensive gene panel. We demonstrated that sequencing paired tumor DNA was helpful to interpret data and confirm ctDNA, and thus increased the ratio of patients with detectable ctDNA. This approach might be feasible for mutation analysis of ctDNA in routine diagnostic practice, especially in case of suboptimal plasma quality and quantity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: Several maternal susceptibility loci for preeclampsia (PE) have been discovered amongst Icelandic, Australian/New Zealand, Dutch and Finnish family cohorts, implicating locus heterogeneity. Through candidate gene studies, allele-specific heterogeneity in different populations is also evident. It is therefore likely that numerous population specific PE susceptibility variants exist, differing in their effect size. Despite on-going efforts to identify susceptibility genes for PE, the causal genetic variants still remain obscure., Objectives: The aim of this study is to interrogate the genetic architecture of PE susceptibility by performing a genome-wide linkage scan in a novel familial cohort from Norway., Methods: A total of 480 DNA samples from The Norwegian PE Family Biobank were genotyped at Genomic Core Facility at NTNU. Genome-wide genotyping was performed with the Infinium HumanExome BeadChip (>240,000 markers) (Illumina, USA) that delivers focused coverage of exonic regions of the human genome., Results: A total of 137 families are represented with 222 women with a valid PE diagnosis (SBP⩾140mmHg DBP⩾90mmHg, ⩾2 measurements at least 4h apart with documented proteinuria at ⩾2 occasions occurring after 20weeks of pregnancy), 44 with self-reported PE and 72 women with a healthy pregnancy. The genotyping has just recently been completed with an average call rate of 99.96%. Data and statistical analysis is now underway using MERLIN, R and SOLAR. A description of the Norwegian PE familial cohort plus preliminary results will be presented at the Congress., Conclusion: To our knowledge this is the first SNP-based genome-wide linkage study on PE, and the first performed in a novel Norwegian PE family cohort. By using an approach focusing on functionally relevant markers we anticipate the identification of susceptibility loci that are of substantial importance for disease development., (Copyright © 2013. Published by Elsevier B.V.)

Evidence is conflicting as to the impact of elevated levels of uncoupling protein-2 (UCP-2) on insulin-producing beta cells. Here we investigated effects of a fourfold induction of UCP-2 protein primarily on mitochondrial parameters and tested for replication of positive findings at a lower level of induction. We transfected INS-1 cells to obtain a tet-on inducible cell line. A 48 h exposure to 1 μg/ml of doxycycline (dox) induced UCP-2 fourfold (424 ± 113%, mean±SEM) and 0.1 μg/ml twofold (178 ± 29%, n=3). Fourfold induced cells displayed normal viability (MTT, apoptosis), normal cellular insulin contents and, glucose-induced insulin secretion (+27 ± 11%) as well as D-[U-(14)C]-glucose oxidation (+5 ± 9% at 11 mM glucose). Oxidation of [1-(14)C]-oleate was increased from 4088 to 5797 fmol/μg prot/2h at 3.3mM glucose, p<0.03. Oxidation of L-[(14)C(U)]-glutamine was unaffected. Induction of UCP-2 did not significantly affect measures of mitochondrial membrane potential (Rhodamine 123) or mitochondrial mass (Mitotracker Green) and did not affect ATP levels. Oligomycin-inhibited oxygen consumption (a measure of mitochondrial uncoupling) was marginally increased, the effect being significant in comparison with dox-only treated cells, p<0.05. Oxygen radicals, assessed by dichlorofluorescin diacetate, were decreased by 30%, p<0.025. Testing for the lower level of UCP-2 induction did not reproduce any of the positive findings. A fourfold induction of UCP-2 was required to exert minor metabolic effects. These findings question an impact of moderately elevated UCP-2 levels in beta cells as seen in diabetes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The PA promoter in the human uracil-DNA glycosylase gene (UNG) directs expression of the nuclear form (UNG2) of UNG proteins. Using a combination of promoter deletion and mutation analyses, and transient transfection of HeLa cells, we show that repressor and derepressor activities are contained within the region of DNA marked by PA. Footprinting analysis and electrophoretic mobility shift assays of PA and putative AP-2 binding regions with HeLa cell nuclear extract and recombinant AP-2alpha protein indicate that AP-2 transcription factors are central in the regulated expression of UNG2 mRNA. Chromatin immunoprecipitation with AP-2 antibody demonstrated that endogenous AP-2 binds to the PA promoter in vivo. Overexpression of AP-2alpha, -beta or -gamma all stimulated expression from a PA-luciferase reporter gene construct approximately 3- to 4-fold. Interestingly, an N-terminally truncated AP-2alpha, lacking the activation domain but retaining the DNA binding and dimerization domains, stimulated PA to a level approaching that of full-length AP-2, suggesting that AP-2 overexpression stimulates PA activity by a mechanism involving derepression rather than activation, possibly by neutralizing an inhibitory effect of endogenous AP-2 or AP-2-like factors.

Paraffin-embedded tissue is an important source of material for molecular pathology and genetic investigations. We used DNA isolated from microdissected formalin-fixed, paraffin-embedded gastric tumors for mutation analysis of a region of the human gene for uracil-DNA glycosylase (UNG), encoding the UNG catalytic domain, and detected apparent base substitutions which, after further investigation, proved to be polymerase chain reaction (PCR) artifacts. We demonstrate that low DNA template input in PCR can generate false mutations, mainly guanine to adenine transitions, in a sequence-dependent manner. One such mutation is identical to a mutation previously reported in the UNG gene in human glioma. This phenomenon was not caused by microheterogeneity in the sample material because the same artifact was seen after amplification of a homogenous, diluted plasmid. We did not observe genuine mutations in the UNG gene in 16 samples. Our results demonstrate that caution should be taken when interpreting data from PCR-based analysis of somatic mutations using low amounts of template DNA, and that methods used to enrich putative subpopulations of mutant molecules in a sample material could, in essence, be a further amplification of sequence-dependent PCR-generated artifacts.

The role of uncoupling protein-2 (UCP-2) in beta-cells is presently unclear. We have tested the notion that UCP-2 participates in beta-cell defense against oxidants. Expression of the UCP-2 gene in clonal beta-cells (INS-1) was decreased by 45% after 48 h of culture with vitamin E and selenite. When INS-1 cells were exposed to 200 microM H(2)O(2) for 5 min, the cell viability (MTT assay) decreased to 85 +/- 1, 61 +/- 1, 40 +/- 2, and 39 +/- 2% of control when measured respectively 30 min, 2 h, 6 h, and 16 h after H(2)O(2) exposure. At corresponding time points UCP-2 mRNA levels were 1.01 +/- 0.09, 1.53 +/- 0.15 (P < 0.05), 1.44 +/- 0.18 (P = 0.06), and 1.12 +/- 0.09 fold of control, i.e., transiently increased. We next tested whether overexpression of UCP-2 could enhance resistance of beta-cells toward H(2)O(2) toxicity. A cotransfection method using EGFP as a suitable marker and a human cDNA UCP-2 construct was used for transient overexpression of UCP-2. Transfected cells expressed the gene about 30-fold more than normal cells. After exposure to H(2)O(2) (200 micrometer, 5 min), the survival of UCP-2 overexpressing cells was measured 30-45 min later by flow cytometry. Survival was 13 +/- 0.05% higher than control (EGFP only) cells, P < 0.004 for difference. The results indicate that oxidative stress induces UCP-2 expression in beta-cells, and that UCP-2 serves a role in beta-cell defense against oxidative stress., (Copyright 2001 Academic Press.)

We studied the repair of cyclobutane pyrimidine dimers (CPDs) in the 5' terminal part of the transcriptionally inactive O6-methylguanine-DNA methyltransferase (MGMT) gene of MGMT-deficient human cell lines (A172, A-253 and WI-38 VA13) and in a proficient cell line (HaCaT), in which the MGMT gene was transcribed. Repair rates in the MGMT gene were compared with those in the active uracil-DNA glycosylase (UNG) and c-myc genes, and those in the repressed X-linked 754 locus and the RNA polymerase I-transcribed ribosomal gene cluster. In the active MGMT gene, there was a distinct strand specificity with more repair in the template (transcribed) strand (TS) than in the non-template strand (NTS). In contrast, no apparent strand bias in the repair of CPDs was observed in the inactive MGMT gene in the MGMT deficient cell lines, although the rates of repair varied between different cell lines. Repair in the inactive MGMT gene was consistently lower than repair in the NTSs of the expressed genes, and approached the generally poor repair of the repressed 754 locus. Whereas repair in the UNG gene was strand-specific in HaCaT, A-172 and WI-38 VA13 cells, no clear strand bias in repair of this gene was evident in A253 cells and repair was relatively inefficient. Although the repair kinetics was essentially similar in the two strands of the c-myc gene in all cell lines examined, the rate and extent of repair were in general significant, probably due to an observed transcription of both strands in the c-myc region. In conclusion, our results indicate that the relative rates of repair in inactive MGMT genes are comparable to those of repressed loci and are lower than repair rates in the NTSs of active genes, but the absolute rate of repair varies between different transformed cells.

We have studied the effect of low levels of paracetamol (0.3 and 1.0 mM) on gene-specific DNA repair, recovery of total RNA synthesis and cytotoxicity after exposure of human keratinocyte cells (HaCaT) to ultraviolet (UV) irradiation. Repair of cyclobutane pyrimidine dimers (CPDs) was measured in the transcriptionally active uracil-DNA glycosylase (UNG) and c-MYC loci. Repair of both strands in the UNG gene was consistently lower in the presence of paracetamol, but this reduction reached significance only at 8 h after irradiation and no dose-response was observed. For the c-MYC gene, we found no significant effect of paracetamol on the repair of CPDs, possibly because UV-irradiation is known to induce transcription of the c-MYC gene and enhanced transcription coupled repair might counteract a negative effect of paracetamol on global genome repair. A dose-dependent delay in the recovery of total RNA synthesis after UV exposure was observed in the presence of paracetamol, which also caused a 20% increase in UV-induced cytotoxicity after 24 h. Paracetamol had no significant effect on either RNA synthesis or cell survival in the absence of UV after 24 h, but reduced cell survival by approximately 10% (at 0.3 mM) and 50%, (at 1.0 mM) after 96 h exposure. Our results demonstrate that paracetamol may inhibit gene-specific repair of CPDs by affecting global genome repair and that different genes may be differentially affected.