Monitoring humans for somatic mutation in the endogenous pig‐a gene using red blood cells

Abstract

The endogenous X-linked PIG-A gene is involved in the synthesis of glycosyl phosphatidyl inositol (GPI) anchors that tether specific protein markers to the exterior of mammalian cell cytoplasmic membranes. Earlier studies in rodent models indicate that Pig-a mutant red blood cells (RBCs) can be induced in animals treated with genotoxic agents, and that flow cytometry can be used to identify rare RBCs deficient in the GPI-anchored protein, CD59, as a marker of Pig-a gene mutation. We investigated if a similar approach could be used for detecting gene mutation in humans. We first determined the frequency of spontaneous CD59-deficient RBCs (presumed PIG-A mutants) in 97 self-identified healthy volunteers. For most subjects, the frequency of CD59-deficient RBCs was low (average of 5.1 ± 4.9 × 10(-6) ; median of 3.8 × 10(-6) and mutant frequency less than 8 × 10(-6) for 75% of subjects), with a statistically significant difference in median mutant frequencies between males and females. PIG-A RBC mutant frequency displayed poor correlation with the age and no correlation with the smoking status of the subjects. Also, two individuals had markedly increased CD59-deficient RBC frequencies of ∼300 × 10(-6) and ∼100 × 10(-6) . We then monitored PIG-A mutation in 10 newly diagnosed cancer patients undergoing chemotherapy with known genotoxic drugs. The frequency of CD59-deficient RBCs in the blood of the patients was measured before the start of chemotherapy and three times over a period of ∼6 months while on/after chemotherapy. Responses were generally weak, most observations being less than the median mutant frequency for both males and females; the greatest response was an approximate three-fold increase in the frequency of CD59-deficient RBCs in one patient treated with a combination of cisplatin and etoposide. These results suggest that the RBC PIG-A assay can be adopted to measuring somatic cell mutation in humans. Further research is necessary to determine the assay's sensitivity in detecting mutations induced by genotoxic agents acting via different mechanisms.

The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup

“…In fact, approaches for scoring Pig-a/PIG-A mutant cells have been described for all these species [1][2][3][13][14][15][16]. Although credit for the original flow cytometric methodology for measuring mutant cells goes to David Araten and Lucio Luzzatto, who described PIG-A assays for human granulocytes and RBCs [17], human and monkey assays are now much less well developed than the rat assay.…”

“…Mammalian species used in other toxicological/investigational studies are amenable for use, theoretically, and there are a number of publications on Pig-a (or PIG-A) assays using other species (e.g., human [15,17], mouse [2,13]). However, a recommendation for their routine use in safety assessment studies cannot be made at this time because standard protocols have not yet been evaluated extensively or published.…”

“…Also, the human PIG-A assay may have value in clinical settings, where Pig-a assays conducted in laboratory animals could provide a translational biomarker for endogenous mutation in vivo. For example, the Pig-a and PIG-A assays could be used for monitoring the long-term effects of genotoxic chemotherapy [15] and aid in estimating the likelihood of developing secondary malignancies. Additionally, the PIG-A assay could be used in epidemiology studies for monitoring the health status (mutation accumulation) in populations exposed to potentially adverse environments, including occupational or accidental exposures to hazardous chemicals.…”

The Human RBC PIG-A Gene Mutation Assay

“…We have built upon Araten's approach for analysis of mutant RBCs, modified the labeling protocol and sample analysis strategy (presented in the following section of this chapter in greater detail), and determined the frequency of CD59-deficient, presumed PIG-A mutants, in the blood of a large number of untreated self-identified healthy individuals of both sexes and from various ethnic backgrounds. We also have measured RBC PIG-A mutant frequencies in newly diagnosed cancer patients before the start of prescribed chemotherapy with genotoxic antineoplastic drugs and at several time points while on or post-chemotherapy [16].…”

Both PIGA and PIGL mutations cause GPI‐a deficient isolates in the Tk6 cell line

“…Currently, there is great interest in the development of flow cytometry-based assays to determine the loss of function of cell membrane glycosylphosphatidylinositols for mutagenicity testing of chemicals in rodents and monitoring of exposures in humans [Dertinger and Heflich 2011; Dertinger et al 2011; Dobrovolsky et al 2011; Sadiq et al 2012; Dobrovolsky et al 2013; Horibata et al 2013; Kimoto et al 2013; Dertinger et al 2014; Kruger et al 2014; Onami et al 2014]. Loss of surface proteins maintained by GPI-a’s is conventionally assessed by flow cytometry or by resistance (lack of cell killing) to the toxin aerolysin.…”