Analysis of mutation in the rat Pig-a assay: II. Studies with bone marrow granulocytes

The in vivo erythrocyte Pig-a gene mutation assay measures the phenotypic loss of GPI-anchored surface markers. Molecular analysis of the marker-deficient erythrocytes cannot provide direct proof that the mutant phenotype is due to mutation in the Pig-a gene because mammalian erythrocytes lack genomic DNA. Granulocytes are nucleated cells that originate from myeloid progenitor cells in bone marrow as is the case for erythrocytes, and thus analysis of Pig-a mutation in bone marrow granulocytes can provide information about the source of mutations detected in the erythrocyte Pig-a assay. We developed a flow cytometric Pig-a assay for bone marrow granulocytes and evaluated granulocyte Pig-a mutant frequencies in bone marrow from male rats treated acutely with N-ethyl-N-nitrosourea (ENU). Bone marrow cells from these rats were stained with anti-CD11b for identifying granulocytes and anti-CD48 for detecting the Pig-a mutant phenotype. The average Pig-a mutant frequency in granulocyte precursors of control rats was 8.42 × 10^-6 , whereas in ENU-treated rats it was 567.13 × 10^-6 . CD11b-positive/CD48-deficient mutant cells were enriched using magnetic separation and sorted into small pools for sequencing. While there were no Pig-a mutations found in sorted CD48-positive wild-type cells, Pig-a mutations were detected in mutant granulocyte precursors. The most frequent mutation observed was T→A transversion, followed by T→C transition and T→G transversion, with the mutated T on the nontranscribed DNA strand. While the spectrum of mutations in bone marrow granulocytes was similar to that of erythroid cells, different Pig-a mutations were found in mutant-phenotype granulocytes and erythroids from the same bone marrow samples, suggesting that most Pig-a mutations were induced in bone marrow cells after commitment to either the granulocyte or erythroid developmental pathway. Environ. Mol. Mutagen. 59:733-741, 2018. © 2018 Wiley Periodicals, Inc.