Kari Bente Foss Haug

The human ABC transporter pseudogene family: Evidence for transcription and gene-pseudogene interference

BackgroundPseudogenes are an integral component of the human genome. Little attention, however, has so far been paid to the phenomenon that some pseudogenes are transcriptionally active. Recently, we demonstrated that the human ortholog of the rodent testis-specific ATP-binding cassette (ABC) transporter Abca17 is a ubiquitously transcribed pseudogene (ABCA17P). The aim of the present study was to establish a complete inventory of all ABC transporter pseudogenes in the human genome and to identify transcriptionally active ABC transporter pseudogenes. Moreover, we tested the hypothesis that a regulatory interdependency exists between ABC transporter pseudogenes and their parental protein coding equivalents.ResultsSystematic bioinformatic analysis revealed the existence of 22 ABC transporter pseudogenes within the human genome. We identified two clusters on chromosomes 15 and 16, respectively, which harbor almost half of all pseudogenes (n = 10). Available information from EST and mRNA databases and RT-PCR expression profiling indicate that a large portion of the ABC transporter pseudogenes (45%, n = 10) are transcriptionally active and some of them are expressed as alternative splice variants. We demonstrate that both pseudogenes of the pseudoxanthoma elasticum gene ABCC6, ABCC6P1 and ABCC6P2, are transcribed. ABCC6P1 and ABCC6 possess near-identical promoter sequences and their tissue-specific expression profiles are strikingly similar raising the possibility that they form a gene-pseudogene dual transcription unit. Intriguingly, targeted knockdown of the transcribed pseudogene ABCC6P1 resulted in a significant reduction of ABCC6 mRNA expression levels.ConclusionThe human genome contains a surprisingly small number of ABC transporter pseudogenes relative to other known gene families. They are unevenly distributed across the chromosomes. Importantly, a significant portion of the ABC transporter pseudogenes is transcriptionally active. The downregulation of ABCC6 mRNA levels by targeted suppression of the expression of its pseudogene ABCC6P1 provides evidence, for the first time, for a regulatory interdependence of a transcribed pseudogene and its protein coding counterpart in the human genome.

Global miRNA expression analysis of serous and clear cell ovarian carcinomas identifies differentially expressed miRNAs including miR-200c-3p as a prognostic marker

BackgroundImproved insight into the molecular characteristics of the different ovarian cancer subgroups is needed for developing a more individualized and optimized treatment regimen. The aim of this study was to a) identify differentially expressed miRNAs in high-grade serous ovarian carcinoma (HGSC), clear cell ovarian carcinoma (CCC) and ovarian surface epithelium (OSE), b) evaluate selected miRNAs for association with clinical parameters including survival and c) map miRNA-mRNA interactions.MethodsDifferences in miRNA expression between HGSC, CCC and OSE were analyzed by global miRNA expression profiling (Affymetrix GeneChip miRNA 2.0 Arrays, n = 12, 9 and 9, respectively), validated by RT-qPCR (n = 35, 19 and 9, respectively), and evaluated for associations with clinical parameters. For HGSC, differentially expressed miRNAs were linked to differentially expressed mRNAs identified previously.ResultsDifferentially expressed miRNAs (n = 78) between HGSC, CCC and OSE were identified (FDR < 0.01%), of which 18 were validated (p < 0.01) using RT-qPCR in an extended cohort. Compared with OSE, miR-205-5p was the most overexpressed miRNA in HGSC. miR-200 family members and miR-182-5p were the most overexpressed in HGSC and CCC compared with OSE, whereas miR-383 was the most underexpressed. miR-205-5p and miR-200 members target epithelial-mesenchymal transition (EMT) regulators, apparently being important in tumor progression. miR-509-3-5p, miR-509-5p, miR-509-3p and miR-510 were among the strongest differentiators between HGSC and CCC, all being significantly overexpressed in CCC compared with HGSC. High miR-200c-3p expression was associated with poor progression-free (p = 0.031) and overall (p = 0.026) survival in HGSC patients. Interacting miRNA and mRNA targets, including those of a TP53-related pathway presented previously, were identified in HGSC.ConclusionsSeveral miRNAs differentially expressed between HGSC, CCC and OSE have been identified, suggesting a carcinogenetic role for these miRNAs. miR-200 family members, targeting EMT drivers, were mostly overexpressed in both subgroups, among which miR-200c-3p was associated with survival in HGSC patients. A set of miRNAs differentiates CCC from HGSC, of which miR-509-3-5p and miR-509-5p are the strongest classifiers. Several interactions between miRNAs and mRNAs in HGSC were mapped.

Identification of Genes Particularly Sensitive to Lipopolysaccharide (LPS) in Human Monocytes Induced by Wild-Type versus LPS-Deficient Neisseria meningitidis Strains

ABSTRACT Lipopolysaccharide (LPS) in the outer membrane of Neisseria meningitidis plays a dominant role as an inflammation-inducing molecule in meningococcal disease. We have used microarray analysis to study the global gene expression after exposure of human monocytes for 3 h to wild-type N. meningitidis (106), LPS-deficient N. meningitidis (106 and 108), and purified N. meningitidis LPS (1 ng [33 endotoxin units]/ml) to identify LPS-inducible genes. Wild-type N. meningitidis (106) induced 4,689 differentially expressed genes, compared with 72 differentially expressed genes induced by 106 LPS-deficient N. meningitidis organisms. However, 108 LPS-deficient N. meningitidis organisms induced 3,905 genes, indicating a dose-response behavior of non-LPS cell wall molecules. A comparison of the gene expression patterns from 106 wild-type N. meningitidis and 108 LPS-deficient N. meningitidis organisms showed that 2,401 genes in human monocytes were not strictly LPS dependent. A list of “particularly LPS-sensitive” genes (2,288), differentially induced by 106 wild-type N. meningitidis but not by 108 LPS-deficient N. meningitidis organisms, showed an early expression of beta interferon (IFN-β), most likely through the Toll-like receptor-MyD88-independent pathway. Subsequently, IFN-β may activate the type I IFN signaling pathway, and an unknown number of IFN-β-inducible genes, such as those for CXCL9, CXCL10, CXCL11, IFIT1, IFIT2, IFIT3, and IFIT5, are transcribed. Supporting this, human monocytes secreted significantly higher levels of CXCL10 and CXCL11 when stimulated by 106 wild-type N. meningitidis organisms than when stimulated by 108 LPS-deficient N. meningitidis organisms. Plasma CXCL10, but not CXCL11, was positively correlated (r = 0.67; P < 0.01) to LPS in patients (n = 24) with systemic meningococcal disease. Thus, new circulating biomarkers in meningococcal disease may be suggested through LPS-induced gene expression changes in human monocytes.

Genetic Variation of VKORC1 and CYP4F2 Genes Related to Warfarin Maintenance Dose in Patients with Myocardial Infarction

The aim of this study was to investigate whether the VKORC1*3 (rs7294/9041 G > A), VKORC1*4 (rs17708472/6009 C > T), and CYP4F2 (rs2108622/1347 C > T) polymorphisms were associated with elevated warfarin maintenance dose requirements in patients with myocardial infarction (n = 105) from the Warfarin Aspirin Reinfarction Study (WARIS-II). We found significant associations between elevated warfarin dose requirements and VKORC1*3 and VKORC1*4 polymorphisms (P = .001 and P = .004, resp.), whereas CYP4F2 (1347 C > T) showed a weak association on higher warfarin dose requirements (P = .09). However, analysing these variant alleles in a regression analysis together with our previously reported data on VKORC1*2, CYP2C9*2 and CYP2C9*3 polymorphisms, gave no significant associations for neither VKORC1*3, VKORC1*4 nor CYP4F2 (1347 C > T). In conclusion, in patients with myocardial infarction, the individual contribution to warfarin dose requirements from VKORC1*3, VKORC1*4, and CYP4F2 (1347 C > T) polymorphisms was negligible. Our results indicate that pharmacogenetic testing for VKORC1*2, CYP2C9*2 and CYP2C9*3 is more informative regarding warfarin dose requirements than testing for VKORC1*3, VKORC1*4, and CYP4F2 (1347 C > T) polymorphisms.

Warfarin dose and INR related to genotypes of CYP2C9 and VKORC1 in patients with myocardial infarction

BackgroundWarfarin treatment has a narrow therapeutic range, requiring meticulous monitoring and dosage titration. Individual dosage requirement has recently partly been explained by genetic variation of the warfarin metabolizing enzyme CYP2C9 and the Vitamin K-activating enzyme VKORC1. In the WARIS-II study, comparing three different antithrombotic regimens after myocardial infarction, warfarin treatment reduced thrombotic events, but was associated with more frequent bleeding than use of acetylsalisylic acid (ASA) alone.AimsThe primary aim of the present study was to investigate the relation between genotypes of CYP2C9 and VKORC1 and warfarin maintenance dose in myocardial infarction. The secondary aim was to relate the genotypes to international normalized ratio (INR).MethodsGenotyping was performed in 212 myocardial infarction patients from the WARIS-II study by robotic isolation of DNA from EDTA whole blood (MagNa Pure LC) before PCR amplification (LightCycler) and melting point analysis.ResultsThe 420 C>T substitution of CYP2C9*2, the 1075 A>C substitution of CYP2C9*3 and the 1173 C>T substitution of VKORC1 had minor allele frequencies of, 11.3%, 5.7% and 36.6% respectively. Warfarin weekly dose varied between 17 mg and 74 mg among the patients. INR did not vary between genotypes. Warfarin dosage requirement was significantly associated with CYP2C9 and VKORC1 genotypes, treatment group and age. The VKORC1 genotype contributed 24.5% to the interindividual variation in warfarin dosage, whereas the combined CYP2C9 genotypes were only responsible for 7.2% of the dose variation.ConclusionCYP2C9 and VKORC1 genotype frequencies in myocardial infarction patients appear similar to other patient groups and have similar impact on warfarin maintenance dose.

Serotonin Transporter Polymorphism Modulates N-Back Task Performance and fMRI BOLD Signal Intensity in Healthy Women

Context Exploring intermediate phenotypes within the human brains functional and structural circuitry is a promising approach to explain the relative contributions of genetics, complex behaviors and neural mechanisms in the development of major depressive disorder (MDD). The polymorphic region 5-HTTLPR in the serotonin transporter gene (SLC6A4) has been shown to modulate MDD risk, but the neural underpinnings are incompletely understood. Objective 37 right handed healthy women between 21 and 61 years of age were invited to participate in an fMRI modified n-back study. The functional polymorphism 5-HTTLPR located in the promoter region of the SLC6A4 gene was genotyped using polymerase chain reaction (PCR). Results Short 5-HTTLPR allele carriers showed more blood-oxygen-level-dependent (BOLD) bilateral prefrontal cortex activation in the right [F(2, 30) = 4.8, η2 = .25, p = .026] and left [F(2, 30) = 4.1, η2 = .22, p = .015] inferior frontal gyrus pars triangularis with increasing n-back task difficulty relative to long 5-HTTLPR allele carriers. Short 5-HTTLPR allele carriers had inferior task performance on the most difficult n-back condition [F(2, 30) = 4.9, η2 = .26, p = .014]. Conclusions This activation pattern found in healthy at risk individuals resembles an activation pattern that is typically found in patients suffering from acute MDD. Altered function in these areas may reflect intermediate phenotypes and may help explain the increased risk of depression in short 5-HTTLPR allele carriers.

POLD2 and KSP37 (FGFBP2) correlate strongly with histology, stage and outcome in ovarian carcinomas.

Background Epithelial ovarian cancer (EOC) constitutes more than 90% of ovarian cancers and is associated with high mortality. EOC comprises a heterogeneous group of tumours, and the causes and molecular pathology are essentially unknown. Improved insight into the molecular characteristics of the different subgroups of EOC is urgently needed, and should eventually lead to earlier diagnosis as well as more individualized and effective treatments. Previously, we reported a limited number of mRNAs strongly upregulated in human osteosarcomas and other malignancies, and six were selected to be tested for a possible association with three subgroups of ovarian carcinomas and clinical parameters. Methodology/Principal Findings The six selected mRNAs were quantified by RT-qPCR in biopsies from eleven poorly differentiated serous carcinomas (PDSC, stage III–IV), twelve moderately differentiated serous carcinomas (MDSC, stage III–IV) and eight clear cell carcinomas (CCC, stage I–IV) of the ovary. Superficial scrapings from six normal ovaries (SNO), as well as biopsies from three normal ovaries (BNO) and three benign ovarian cysts (BBOC) were analyzed for comparison. The gene expression level was related to the histological and clinical parameters of human ovarian carcinoma samples. One of the mRNAs, DNA polymerase delta 2 small subunit (POLD2), was increased in average 2.5- to almost 20-fold in MDSC and PDSC, respectively, paralleling the degree of dedifferentiation and concordant with a poor prognosis. Except for POLD2, the serous carcinomas showed a similar transcription profile, being clearly different from CCC. Another mRNA, Killer-specific secretory protein of 37 kDa (KSP37) showed six- to eight-fold higher levels in CCC stage I compared with the more advanced staged carcinomas, and correlated positively with an improved clinical outcome. Conclusions/Significance We have identified two biomarkers which are markedly upregulated in two subgroups of ovarian carcinomas and are also associated with stage and outcome. The results suggest that POLD2 and KSP37 might be potential prognostic biomarkers.

Circulating lipopolysaccharides in the blood from “bioprotein” production workers

Background: Workers producing bacterial single-cell protein (BSCP), “bioprotein,” are exposed to organic dust containing high levels of endoxins (lipopolysaccharides, LPS). Workers in this industry have complained of episodes of fever, fatigue, chest tightness, skin dryness and rubor. The aim of the present study was to quantify LPS and inflammatory mediators in plasma among the workers and non-exposed control subjects. Methods: We included eight non-smoking production workers, aged 32–51 (median 38), and eight non-smoking, non-exposed controls, aged 30–51 (median 39). Airborne and plasma endotoxin concentrations were measured, as well as plasma hsCRP and different cytokines, chemokines and metalloproteinases. Results: The workers who did not use personal respiratory protection were exposed to varying airborne levels of endotoxin, 430 (75–15 000) EU/m3 (median, range). The level of plasma LPS was significantly elevated (p = 0.01) among the workers compared to the non-exposed controls. The workers also had elevated levels of MCP-1 (p = 0.02), MIP-1α (p = 0.05) and MMP-3 (p = 0.04). IL-6 and hsCRP were also elevated among the exposed group, but not significantly (p = 0.10 and p = 0.07, respectively). Conclusions: In this study, we detected LPS in plasma of individuals exposed to high levels of LPS at their workplace. This finding is supported by elevated levels of several inflammatory cytokines among the workers, significantly exceeding that of the non-exposed control group. To the best of our knowledge, this is the first time that plasma LPS, together with increased inflammatory markers in plasma, has been detected in an occupational setting.

The effects of the serotonin transporter polymorphism and age on frontal white matter integrity in healthy adult women.

Studies of populations at genetic risk have the potential to explore the underlying structural and functional mechanisms in the development of psychological disorders. The polymorphic region (5-HTTLPR) in the serotonin transporter gene (SLC6A4) has been associated with major depression (MDD) (Caspi et al., 2003). In healthy women, variation in the human brain white matter microstructure integrity in the uncinate fascicule (UF) has been suggested as an endophenotypes in the development of MDD. Pacheco et al. (2009) found a unique effect of age and 5-HTTLPR within the left frontal UF. The present study examined whether these associations persist along the adult life span. Thirty-seven right-handed healthy women between 21 and 61 years of age were invited for a diffusion MRI study. The functional polymorphism 5-HTTLPR located in the promoter region of the SLC6A4 gene was genotyped using polymerase chain reaction (PCR). Fractional anisotropy (FA) was generated for the UF based on Tract-Based Spatial Statistics (TBSS). Models of emotion regulation circuitry suggest that working memory is important in conscious emotion regulation (Price and Drevets, 2010). To explore if 5-HTTLPR is related to this aspects of emotion processing, a working memory pathway, the superior longitudinal fascicule (SLF) was included. The results demonstrate that age may explain the hypothesized association between 5-HTTLPR and frontal UF white matter integrity in healthy adult women. Both white matter changes associated with the aging process and those associated with growth and development may explain why the earlier reported unique effects of genotype in frontal UF FA do not persist into adulthood.

Quantification of relative changes in specific mRNAs from frozen whole blood - : methodological considerations and clinical implications

Abstract Background: Based on quantification of relative changes in lipopolysaccharide (LPS)-regulated mRNA transcripts, the present study aimed to establish a robotic method to isolate RNA from stabilized frozen whole blood suitable for gene expression analysis. Methods: Whole blood (±LPS) was stored in EasyLyse™ solution or PAXgene® tubes (room temperature and −70°C) for comparison of storage methods, then subjected to robotic isolation of total RNA. Yield, quality and relative changes in 11 selected mRNA transcripts were examined. Method precision (% coefficient of variation) for a longitudinal control was established. The influence of globin mRNA from reticulocytes in quantitative RT-PCR was examined. Results: All storage methods gave a similar high-quality RNA yield. The differences in the 11 specific mRNA quantities stored in EasyLyse or PAXgene® at −70°C were small: mean −0.01 (95% CI –0.19 to 0.17). The CV for mRNAs in the longitudinal control ranged from 3% to 150%. Thus, the number of replicates necessary to detect a 20% difference (power 0.8) ranged from 2–50. Globin mRNA had no influence on quantitative RT-PCR Conclusions: Based on measuring the relative changes in specific mRNAs in LPS-exposed whole blood, we conclude that PAXgene® tubes stored at −70°C could preferentially be used. This may open opportunities for monitoring gene expression changes in clinical settings. Clin Chem Lab Med 2007;45:171–6.