Genetic variation and regulation of oxidative stress related genes

Abstract

Oxidative stress, is a state that has been implicated in the pathogenesis of a variety of human diseases, including atherosclerosis, inflammation, neurodegenerative disease and cancer. In an effort to determine the importance of oxidative stress in disease etiology, by a genetic approach, we have, studied individual variation and regulation of oxidative stress related genes including also basal characterization. When single stranded conformation polymorphism (SSCP) was performed in order to search for new genetic polymorphisms in the microsomal glutathione transferase I (MGST1) gene, it became evident that the laborious task of identifying new polymorphisms would be simplified if variants could be found in silico in the human EST data base. The approach was tested with five known polymorphisms in three different genes. They could all be observed in the EST data base. The same approach was then employed to detect new polymorphisms in antioxidant related genes. Two of 10 indicated variants from EST searches could be identified as common, where one resulted in an amino acid substitution (Pro-Leu) in glutathione peroxidase 1. Further analysis of the phenotypic outcome of this polymorphism, revealed that the amino acid substitution does not alter enzyme activity in red blood cells. In addition, no association between genotype and the risk of suffering stroke was evident.

By direct sequence analysis, an additional polymorphism in the catalase promoter was discovered, a C-T nucleotide substitution located -330 base pairs from the translation start site. This variation was shown to have substantial impact on the basal gene expression as well as on catalase levels in red blood cells. The polymorphism can thus be used to study a possible impact of oxidative stress in disease utilizing genetic epidemiological association studies. Alternative splicing is one of several gene regulatory events that occur in humans. EST analysis of MGST1 transcripts revealed the existence of several first non-coding exons. In order to identify the predominant first exon and thereby the functional promoter, Northern blotting was performed. By comparing mRNA expression probed with full-length cDNA or two of the alternative first exons in several human tissues it was shown that the sequence corresponding to the most abundant EST sequence was predominantly expressed in several tissues. The gene organization of human prostaglandin E synthase was characterized, exhibiting identical exon/intron boundary positions as its closest relative, MGST1. The chromosome localization was determined as well as some regulatory properties. Similarities to the regulation of cyclooxygenase 2 by interleukin 1ß (IL- 1ß), suggested co-ordinate regulation whereas phenobarbital totally repressed PGES transcription. The latter finding is, to our knowledge, the first example of negative gene regulation by this compound.