CD59‐deficient bone marrow erythroid cells from rats treated with procarbazine and propyl‐nitrosourea have mutations in thePig‐agene

Abstract

Procarbazine (PCZ) and N-propyl-N-nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI-anchored marker-deficient mutant-phenotype red blood cells (RBCs) in the flow cytometry-based rat RBC Pig-a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig-a mutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59-deficient mutant-phenotype BMEs from PCZ-and PNU-treated rats and examined their endogenous X-linked Pig-a gene by next generation sequencing. Pig-a mutations consistent with the properties of PCZ and PNU were found in sorted mutant-phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of the Pig-a gene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatmentinduced mutations were distributed across the protein coding sequence of the Pig-a gene. The causal relationship between BMEs and RBCs and the agent-specific mutational spectra in CD59-deicient BMEs indicate that the rat RBC Pig-a assay, scoring CD59-deficient mutant-phenotype RBCs in peripheral blood, detects Pig-a gene mutation. K E Y W O R D S flow cytometry, glycosyl phosphatidylinositol, red blood cells, sequencing 1 | INTRODUCTION Mutation in the mammalian endogenous X-linked Pig-a gene can disrupt glycosyl phosphatidylinositol (GPI) synthesis, resulting in a mutant phenotype that is characterized by a deficiency in specific GPI-anchored surface markers (Kinoshita et al., 1997; Kinoshita, 2014). Since mutation in the human PIG-A gene is associated with a disease, paroxysmal nocturnal hemoglobinuria, diagnostic assays have been designed for detecting mutant-phenotype cells

Pig‐a gene mutations in bone marrow granulocytes of procarbazine‐treated F344 rats

“…Most mutations occurred in protein coding sequences but two were found in canonical intronic splice site regions: animal #8 had a T > C transition in position 784 and animal #18 had a A > T transversion in position #9482. In addition, two mutations were found in intronic regions having no obvious functional significance: animal #7 had a mutation in position #10018 and animal #13 had a G > A at position #9406 of the reference sequence (reference sequence shown in supporting information in Revollo et al (2020)). In addition to the 25 simple base pair substitutions, one mutation was a frameshift, deleting an A:T base pair at position #85 in the coding sequence of the reference genome.…”

“…The main goal of this study was to evaluate the mutagenic potential of PCZ in the rat BMG Pig-a assay and provide further evidence that the phenotypic loss of GPI-anchors from the cell surface is due to mutation in the Pig-a gene. A secondary objective was to compare the spectrum of mutations detected in BMGs with the spectrum in BMEs from the same rats (the BME mutations were described by (Revollo et al, 2020)). This was done to seek further insight into how mutations are induced in the erythrocyte Pig-a gene mutations assay.…”

“…The flushed bone marrow was filtered and diluted with 20 mL of ice-cold PBS + 2% FBS (modified PBS) and divided in half. Half of the cells were used for the erythroid Pig-a assay described by Revollo et al (2020), and the other half was used for the Pig-a assay in BMGs. The diluted bone-marrow cells were further processed for BMG preparation by RBC lysis, first by treatment with a hypotonic (0.2%) saline solution followed immediately by addition of an equal volume of hypertonic (1.8%) saline solution.…”

“…The sequencing reads were de-multiplexed using their indices and reassigned to specific groups of aliquots for each animal. After grouping the data, all the reads were merged into read1 (R1) and read2 (R2) files and then aligned with the Pig-a reference sequence used in Revollo et al (2017) and Revollo et al (2020). Alignment was conducted using the BWA-Mem aligner (Li and Durbin, 2009) available at http://bio-bwa.sourceforge.net/.…”

Recommendations for conducting the rodent erythrocyte Pig‐a assay: A report from the HESI GTTCPig‐a Workgroup

“…The phosphatidylinositol glycan, class A ( Pig‐a ) gene codes for an enzyme that is essential for glycosylphosphatidylinositol (GPI) anchor biosynthesis (Miyata et al ., 1993). Thus, inactivating Pig‐a mutations result in cells that lack cell surface GPI anchors, and as a consequence, GPI‐anchored protein(s); this phenotype represents a reliable reporter of Pig‐a mutation in vivo (Kimoto et al ., 2011b; Revollo et al ., 2018, 2019, 2020; Dad et al ., 2020). The analytical approach used to perform these assays utilizes fluorescently conjugated antibodies against GPI‐anchored cell surface epitopes, which makes it possible to measure mutant cell frequencies via flow cytometry (reviewed by Gollapudi et al ., 2015).…”