Molecular genetic studies of oxidative stress related genes

This work has focused on the basal characterisation of transcriptional regulation, including genetic variants, in oxidative stress related genes and the possible impact of such variation in cardiovascular disease. The genes under study were catalase and MGST1/PGES where the two former protect from hydrogen peroxide and lipid hydroperoxides respectively and the latter is important in e.g. inflammation. Genomic DNA from ten individuals was sequenced resulting in the discovery of a promoter polymorphism at position –262 from the transcription start site in the human catalase gene. The C to T exchange was shown by reporter gene analysis and transient transfection to influence promoter activity. EMSA analysis showed that there is also a difference in transcription factor binding between the two variants. Catalase protein content measured in whole blood by Western Blot revealed that individuals carrying the T allele have significantly higher catalase levels. This result is consistent with the stronger promoter activity of this allelic variant. Regulation of the Catalase gene in humans is still unclear. Deletion analysis of the promoter showed that the sequence between –323 and –134 (from the transcription start site) is required for expression of human catalase in HepG2 cells. This sequence includes several GC and CCAAT boxes. Mutational analysis of these cis-acting elements revealed that one CCAAT box (“4”) was essential for basal expression. EMSA analysis showed that the transcription factor NF-Y binds to this element. The MAPEG members, MGST1 and PGES share 38% identity at the amino acid level and both genes lack TATA boxes. The MGST1 promoter contains one GC box. Reporter constructs transfected into HepG2 cells showed that the GC box is necessary for basal transcription of MGST1. EMSA analysis showed that Sp1 binds to this GC box. The putative promoter of the PGES gene includes two GC boxes. Mutational analysis confirmed that both of them are functional and transfections in A549 cells showed that they are involved in the basal expression of the gene. The transcription factors Sp1 and Sp3 were shown to bind to the GC boxes using EMSA analysis. The catalase –262 C>T polymorphism was analysed with regard to its potential impact on MI. 2343 individuals (cases and controls) from the SHEEP material were genotyped using the Taqman assay. There was no influence of the polymorphism on risk for MI in the general population. However diabetic carriers of the T allele had a significantly lower risk (OR=0.46, 95% CI 0.23-0.89) of MI compared to diabetics with the C allele. It appears that higher levels of catalase are protective in a risk population. In summary the work in this thesis has contributed to the understanding of the basal regulation of oxidative stress related genes and the impact of polymorphism in such genes on transcriptional regulation and disease susceptibility. LIST OF PUBLICATIONS This thesis is based on the following original articles, which will be referred to in the text by their Roman numerals. The published articles are printed with permission. I. Forsberg L, Lyrenas L, de Faire U, Morgenstern R. A common functional CT substitution polymorphism in the promoter region of the human catalase gene influences transcription factor binding, reporter gene transcription and is correlated to blood catalase levels. Free Radic Biol Med. 2001 Mar 1;30(5):500-5. II. Ekstrom L, Lyrenas L, Jakobsson PJ, Morgenstern R, Kelner MJ. Basal expression of the human MAPEG members microsomal glutathione transferase 1 and prostaglandin E synthase genes is mediated by Sp1 and Sp3. Biochim Biophys Acta. 2003 Jun 19;1627(2-3):79-84. III. Lyrenas L, Hagnan C, Morgenstern, R, Ekstrom L. Basal expression of the human catalase gene in HepG2 cells is mediated by NF-Y. Manuscript (2005) IV. Lyrenas L, Bennet AM, de Faire U, Morgenstern R. Catalase gene –262 C>T polymorphism influences the risk of myocardial infarction in diabetic Swedes. Manuscript (2005) PUBLICATIONS NOT INCLUDED IN THE THESIS Lyrenas, L. , Zotova, E. , Ekstrom, L. and Morgenstern, R. : Oxidative stress, genetic variation and disease. In: Oxidative stress, genetic variation and diseaseQuek, J. (Ed.). World Scientific Publishing Co., 2005. InPress. Lyrenas, L. , Wincent, E. , Forsberg, L. and Morgenstern, R. : The Potential Impact of Polymorphism on Oxidative Stress Status. In: Redox-Genome Interactions in Health and Disease. Cadenas, E. (Ed.), Marcel Dekker, Inc., New York, 2004. Bennet AM, Prince JA, Fei GZ, Lyrenas L, Huang Y, Wiman B, Frostegard J, Faire U. Interleukin-6 serum levels and genotypes influence the risk for myocardial infarction. Atherosclerosis. 2003 Dec;171(2):359-67.