Human erythrocyte PIG‐A assay: An easily monitored index of gene mutation requiring low volume blood samples

Abstract

This laboratory has previously described a method for scoring the incidence of rodent blood Pig-a mutant phenotype erythrocytes using immunomag-netic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends this approach to human blood. The frequencies of CD59- and CD55-negative reticulo-cytes (RETCD59−/CD55−) and erythrocytes (RBCCD59−/CD55−) seve as phenotypic reporters of PIG-A gene mutation. Immunomagnetic separation was found to provide an effective means of increasing the number of reticulocytes and erythro-cytes evaluated. Technical replicates were utilized to provide a sufficient number of cells for precise scoring while at the same time controlling for procedural accuracy by allowing comparison of replicate values. Cold whole blood samples could be held for at least one week without affecting reticulo-cyte, RETCD59−/CD55− or RBCCD59−/CD55− frequencies. Specimens from a total of 52 nonsmoking, self-reported healthy adult subjects were evaluated. The mean frequency of RETCD59−/CD55− and RBCCD592−/CD55− were 6.0 × 10−6 and 2.9 × 10−6, respectively. The difference is consistent with a modest selective pressure against mutant phenotype erythrocytes in the circulation, and suggests advantages of studying both populations of erythrocytes. Whereas intra-subject variability was low, inter-subject variability was relatively high, with RETCD59−/CD55− frequencies differing by more than 30-fold. There was an apparent correlation between age and mutant cell frequencies. Taken together, the results indicate that the frequency of human PIG-A mutant phenotype cells can be efficiently and reliably estimated using a labeling and analysis protocol that is well established for rodent-based studies. The applicability of the assay across species, its simplicity and statistical power, and the relatively non-invasive nature of the assay should benefit myriad research areas involving DNA damage, including studies of environmental factors that modify “spontaneous” mutation frequencies.

Differential genotoxicity of acrylamide in the micronucleus andPig-a gene mutation assays in F344 rats and B6C3F1 mice

“…The increase in micronuclei in mice in the present study was highly consistent with results of a previous multiple dose micronucleus assay conducted in our laboratory (21). For the present study of rats and mice exposed to acrylamide we assessed the induction of chromosomal damage by using the in vivo micronucleus assay and mutation of an endogenous gene using a novel flow cytometry based assay that can detect mutation at the Pig-a locus in the blood of mice, rats and humans (38,(48)(49)(50). The Pig-a gene mutation assay is based on the loss of Pig-a, a gene whose product is essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors on the cell surface of reticulocytes and red blood cells (51).…”

A population study using the human erythrocyte PIG‐A assay

“…A limited number of pilot studies have been conducted using the human PIG‐A assay (Dobrovolsky et al ; Rondelli et al ; Dertinger et al ). Dobrovolsky et al () developed a human red blood cell (RBC) PIG‐A assay based on the initial work of Araten et al (), and provided data indicating that the assay could be used to detect somatic cell mutation in humans.…”

“…Dobrovolsky et al () developed a human red blood cell (RBC) PIG‐A assay based on the initial work of Araten et al (), and provided data indicating that the assay could be used to detect somatic cell mutation in humans. Dertinger et al () described methods that used both reticulocytes (RETs) and red blood cells (RBCs) to acquire reliable and reproducible human PIG‐A gene mutant frequencies (MFs). Rondelli et al () performed studies in peripheral blood granulocytes.…”

Le gènePIG-A, nouveau marqueur de mutagenèse

“…Ils permettent donc la réalisation du test PIG-A sur de très petits volumes sanguins 3 . Il faut cependant noter que la fréquence spontanée des érythrocytes GPI-déficients est plus faible que celle des autres populations cellulaires 4 [4,22,23] et que l'érythrocyte étant la cellule mature de la lignée érythroïde, le temps d'expression phénotypique des mutants correspond à la durée d'une érythropoïèse, soit 18 à 20 jours chez l'homme [24]. Les réticulocytes, précurseurs immédiats des érythrocytes, constituent une autre population d'intérêt.…”

“…Le séquençage du gène PIG-A réalisé sur des cellules issues de patients HPN et de sujets sains a ainsi identifié plus de 100 mutations inactivantes (parmi lesquelles des mutations non-sens, faux-sens, des décalages du cadre de lecture, des substitutions, et de larges délétions) aboutissant toutes au même phénotype de déficience en GPI, suggérant que le gène PIG-A puisse être sensible à un grand nombre de modes d'actions génotoxiques [15,34]. Deux lacunes identifiées par l'IWGT en 2015 ont été comblées par la publication de deux études récentes : les performances du test ne dépendent en fait pas du nombre de copies du chromosome X portées par l'individu, les fréquences spontanées et induites de cellules mutées pour le gène PIG-A n'étant pas différentes entre mâles et femelles [23,35,36] ; l'érythropoïèse compensatoire observée après exposition à un agent hémolysant non génotoxique (comme le 2-butoxyéthanol) n'entraîne pas d'augmentation de la fréquence des cellules GPI-déficientes [37]. Ces qualités ont initié l'évaluation du test PIG-A in vitro : le séquen-çage du gène PIG-A réalisé sur des lymphoblastes humains cultivés in vitro et soumis à divers génotoxiques connus, a révélé des résultats comparables à ceux obtenus in vivo chez la souris après exposition aux mêmes agents [38].…”