Confirmation of Pig-a mutation in flow cytometry-identified CD48-deficient T-lymphocytes from F344 rats

Abstract

The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N-ethyl-N-nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect-gene mutation in the Pig-a gene.

CD48‐deficient T‐lymphocytes from DMBA‐treated rats have de novo mutations in the endogenous Pig‐a gene

“…Individual CD48‐deficient T‐cells were sorted using a flow cytometer into culture medium for clonal expansion and subsequent molecular analysis of Pig‐a mutation. We found that the spectrum of Pig‐a mutations in CD48‐deficient T‐cells derived from DMBA‐treated rats was different from the spectrum of Pig‐a mutations in ENU‐treated rats—the former was dominated by A→T transversions, whereas the latter was dominated by T→A transversions [Revollo et al, ]. The nature of the basepair substitutions is consistent with the types of DNA damage produced by the two agents (adducts with dA vs. adducts with dT).…”

“…The relative fraction of the mutant genotype clones is superimposed on the averaged absolute frequency of CD48‐deficient (presumed Pig‐a mutant) T‐cells determined in vehicle‐control and mutagen‐treated F344 male rats. An asterisk represents the data that were reported in an earlier study [Revollo et al, ]. The frequency of clones with WT Pig‐a genotype (false mutant clones) is small relative to the frequency of true Pig‐a mutant clones in ENU‐ and DMBA‐treated rats.…”

“…After stimulation and expansion, CD48 cell‐surface expression in such clones could return to normal, CD48‐positive levels. In our previous study of Pig‐a mutation, we reanalyzed CD48 expression in six clones from one ENU‐treated rat [Revollo et al, ]. These clones had developed from sorted CD48‐deficient “mutant phenotype” cells after 12 days of culture, and in one of these clones the level of CD48 expression was characteristic of WT T‐cells (this particular clone was not analyzed for Pig‐a mutation).…”

“…The complete first exon and part of the second exon of the Pig‐a gene are nontranslated. Black triangles “▸” represent primers employed in this study for amplification and sequencing of Pig‐a exons from genomic DNA, and in the previous study [Revollo et al, ] for Pig‐a cDNA synthesis and amplification/sequencing of its fragments. The positions of the primers relative to Pig‐a genomic DNA and mRNA/cDNA are shown in their 5′ to 3′ prime orientations.…”

Suitability of Long‐Term Frozen Rat Blood Samples for the Interrogation of Pig‐a Gene Mutation by Flow Cytometry

“…For instance, in the case of rat erythrocytes, fluorochrome‐conjugated antibodies against the GPI‐anchored antigen CD59, together with flow cytometric analysis, are used to score the incidence of nonfluorescent mutant cells relative to fluorescent wild‐type cells. DNA‐sequencing results clearly support the use of the GPI anchor‐deficient phenotype as a reliable reporter of Pig‐a gene mutation (Kimoto et al, ; Dobrovolsky et al, ; Nicklas et al, ; Revollo et al, , ; Krüger et al, ; Bemis et al, ).…”