Lena Forsberg

Human glutathione dependent prostaglandin E synthase: gene structure and regulation

A P1 clone containing the gene for human glutathione dependent PGE synthase (PGES) was isolated and characterized. The gene is divided into three exons, spans 14.8 kb and was localized to chromosome 9q34.3. In A549 cells, the protein and activity levels of PGES were increased by interleukin‐1β. This increase was prevented by phenobarbital. Reporter constructs containing the 5′‐flanking region of exon 1, which exhibited strong promoter activity, responded accordingly, except that interleukin‐1β induced a transient increase followed by a decrease. As cyclooxygenase 2 expression has been reported to respond in a similar fashion, a transcriptional regulatory basis for the observed co‐regulation with PGES is implied. The strong down‐regulation by phenobarbital raises important issues concerning its mechanisms of action.

Direct analysis of single‐nucleotide polymorphism on double‐stranded DNA by pyrosequencing

Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single‐nucleotide polymorphism on double‐stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template‐preparation steps is discussed.

Low yield of polymorphisms from EST blast searching: Analysis of genes related to oxidative stress and verification of the P197L polymorphism in GPX1

To determine new polymorphisms in the antioxidant enzymes superoxide dismutase, glutathione peroxidases, catalase, and microsomal glutathione transferase 1, a search of the human expressed sequence tags (EST) database was performed (with BLAST 2.0). When any mutation, indicated by the BLAST search, gave rise to a nonconservative amino acid change we performed polymerase chain reaction (PCR) restriction analysis and/or sequence analysis of genomic DNA from human subjects in order to verify these potential polymorphisms. Of nine indicated polymorphisms from the EST analysis found in four different antioxidant enzymes, we could verify one, an amino acid substitution Pro‐Leu at amino acid position 197 (P197L), in the glutathione peroxidase 1 gene. The corresponding allele frequencies were ≈70/30%. In addition, a silent mutation (1167T/C) in the catalase gene indicated by the BLAST search could also be verified. Six to nine individuals were analyzed per indicated polymorphism, so that only common polymorphisms would be found. The indicated mutations not verified by direct analysis thus cannot be excluded as allelic variation in the human population. These results show that the EST database can be used to search for polymorphisms in genes with high abundance in the human EST database. In addition to the EST analysis, PCR/single‐strand conformation polymorphism (SSCP) was employed for the analysis of the microsomal glutathione transferase 1 gene. No polymorphism in the coding sequence could be detected in the gene by either method. The high degree of conservation of the microsomal glutathione transferase 1 gene indicates an important physiological function for this enzyme. Hum Mutat 13:294–300, 1999.

Repeated blood pressure measurements in a sample of Swedish twins: heritabilities and associations with polymorphisms in the renin-angiotensin-aldosterone system.

Background Twin and family studies have shown that genetic effects explain a relatively high amount of the phenotypic variation in blood pressure. However, many studies have not been able to replicate findings of association between specific polymorphisms and diastolic and systolic blood pressure. Methods In a structural equation-modelling framework the authors investigated longitudinal changes in repeated measures of blood pressures in a sample of 298 like-sexed twin pairs from the population-based Swedish Twin Registry. Also examined was the association between blood pressure and polymorphisms in the angiotensin-I converting enzyme and the angiotensin II receptor type 1 with the ‘Fulker’ test. Both linkage and association were tested simultaneously revealing whether the polymorphism is a Quantitative Trait Locus (QTL) or in linkage disequilibrium with the QTL. Results Genetic influences explained up to 46% of the phenotypic variance in diastolic and 63% of the phenotypic variance in systolic blood pressure. Genetic influences were stable over time and contributed up to 78% of the phenotypic correlation in both diastolic and systolic blood pressure. Non-shared environmental effects were characterised by time specific influences and little transmission from one time point to the next. There was no significant linkage and association between the polymorphisms and blood pressure. Conclusions There is a considerable genetic stability in both diastolic and systolic blood pressure for a 6-year period of time in adult life. Non-shared environmental influences have a small long-term effect. Although associations with the polymorphisms could not be replicated, results should be interpreted with caution due to power considerations.

Identification of genetic polymorphisms in the 'expressed sequence tag' (EST) database

▼Genetic variation in the human population has beenstudied to gain insight into the functional importance ofspecific genes in disease and drug metabolism. For ex-ample, polymorphic variants of genes that are importantin the regulation of blood pressure, blood clotting andblood lipids are studied in relation to cardiovascular dis-ease (Ref. 1). The relation of detoxification enzymes to can-cer risk (Ref. 2, 3, 4, 5, 6, 7) and idiosyncratic drug effects(Ref. 8, 9), the latter with the aim to obtain individual-ized drug-therapy regimens, are the focus of world-wide ef-forts. Individual genotyping can now be performed quicklyand easily by PCR-based methods utilizing minute quan-tities of DNA. However, finding new unknown polymor-phisms in a gene is a tedious task involving either a searchfor restriction fragment length polymorphisms (RFLPs) onSouthern blots or PCR amplifications of suitably sized seg-mentsofthegene,followedbysingle-strand-conformation-polymorphism (SSCP) analysis or direct DNA-sequence de-termination. Because of the considerable interest in, andplans of, cataloguing human DNA sequence variation (Ref.10), improved methodology, including minisequencingstrategies, multiplex reverse dot blots, DNA chips and aTaqMan approach is being developed. However, we showhere that a considerable amount of human DNA sequencevariants can be found