Molecular analysis of GPI-anchor biosynthesis pathway genes in rat strains used for the Pig-a gene mutation assay.

ABSTRACT

Recent studies support the assumption that mutation of the X-linked Pig-a gene is most likely responsible for the mutant phenotype of the cells deficient in glycosylphosphatidylinositol (GPI)-anchored proteins quantified in the rodent Pig-a gene mutation assay. In humans, however, mutations in both alleles of one of the 30 other genes involved in GPI-anchor synthesis, e.g., PIG-L and PIG-O, cause reduced expression of surface GPI-anchored proteins. Here, we investigated the possibility that the loss of the GPI-anchor detected by the rat Pig-a assay also could be caused by mutation in other GPI-biosynthesis genes. 31 samples were obtained from 8 inbred and outbred rat strains commonly used for genetic toxicology assays. In order to investigate possible sources of variation in the Pig-a assay, variant DNA sequences were evaluated in Cd59 and 24 GPI-biosynthesis genes. In some genes, such as Pig-n and Pig-u, homozygous variations occurred in all animals, suggesting that these variations are due to deviations in the reference genome. Heterozygous Pig-s, Pig-w, Pig-o, Pig-c, Pgap1, Pgap2, Pig-k and Pig-t variations were found, however, indicating that these genes could serve as targets for mutation in the assay. Protein alignment for these altered genes was conducted with possible human, mouse and rat phenotypic mutants from the literature; this analysis demonstrated that many of the variations that we detected were in non-conserved sequences and that no phenotypes for any of these variants could be inferred from known mutants from the literature. All heterozygous variants were in outbred rats. Overall, the findings of this study cannot totally rule out the possibility that mutations in GPI-biosynthesis genes other than Pig-a are detected in the Pig-a assay, but suggest that if it occurs, it must occur only rarely and therefore mutations in genes other than Pig-a have little impact on rat-based experiments.