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 dismut ase, glutathione peroxidases, catalase, and microsomal glutathione transfer ase 1, a search of the human expressed sequence tags (EST) database was per formed (with BLAST 2.0), When any mutation, indicated by the BLAST search, gave rise to a nonconservative amino acid change we performed polymerase ch ain 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 differe nt antioxidant enzymes, we could verify one, an amino acid substitution Pro Leu at amino acid position 197 (P197L), in the glutathione peroxidase 1 ge ne. The corresponding allele frequencies were approximate to 70/30%. In add ition, a silent mutation (1167T/C) in the catalase gene indicated by the BL AST search could also be verified. Six to nine individuals were analyzed pe r indicated polymorphism, so that only common polymorphisms would be found. The indicated mutations not verified by direct analysis thus cannot be exc luded as allelic variation in the human population. These results show that the EST database can be used to search for polymorphisms in genes with hig h abundance in the human EST database. In addition to the EST analysis, PCR /single-strand conformation polymorphism (SSCP) was employed for the analys is of the microsomal glutathione transferase 1 gene. No polymorphism in the coding sequence could be detected in the gene by either method. The high d egree of conservation of the microsomal glutathione transferase 1 gene indi cates an important physiological function for this enzyme. Hum Mutat 13:294 -300, 1999. (C) 1999 Wiley-Liss, Inc.