October’s Editor’s Choice article is “CD59-deficient bone marrow erythroid cells from rats treated with procarbazine and propyl-nitrosourea have mutations in the Pig-a gene,” (https://onlinelibrary.wiley.com/doi/10.1002/em.22402) by Javier R. Revollo, Azra Dad, Mason G. Pearce, Roberta A. Mittelstaedt, Andrea Casildo, Rena G. Lapidus, Timothy W. Robinson, and Vasily N. Dobrovolsky.
The PIG-A gene encodes a protein called phosphatidylinositol glycan class A. Cells use this protein to create “anchor” proteins that fasten receptors to the cell surface. Cell surface receptors are needed for a variety of functions, such as receiving signals from the body or interactions with other cells. If the PIG-A gene is mutated – which is to say that it develops a permanent change in its DNA sequence – the consequence is the loss of certain anchor proteins and, subsequently, the expression of the surface receptors that they anchor.
The PIG-A gene is of interest to the medical community because when the gene is mutated in a hematopoietic stem cell (HSC), this causes a rare, life-threatening genetic disease called paroxysmal nocturnal hemoglobinuria (PNH). HSCs live in the bone marrow and are crucial for constantly replenishing the body with white blood cells (cells of the immune system), red blood cells (RBCs), and platelets. When RBCs lack a surface receptor called CD59, which depends on the PIG-A gene for expression, the RBCs are seen by the body as damaged cells and they are destroyed, causing anemia (loss of RBCs), which is a symptom of PNH.
The Pig-a gene mutation assay, which is the focus of the paper by Revollo et al., had its beginnings in the effort to develop a diagnostic test for PNH. A machine called a flow cytometer can be used to detect whether a patient’s RBCs are missing CD59. The patient’s blood is drawn and mixed with an antibody for CD59. A fluorochrome (a chemical that can re-emit light) is attached to the antibody. When the antibody attaches to CD59 receptors, the cells are decorated with the fluorochrome, which can be detected by the flow cytometer. If most of the cells are fluorescent, it is unlikely that the patient has PNH. But if very few cells light up, the patient is missing CD59 and likely has the disease.
Genetic toxicologists, who study the toxic effects of chemicals on DNA, realized that this easy, fast, and inexpensive test could be reimagined as a test to evaluate whether chemical exposures cause mutations. Increases in mutations in general are associated with greater risk for developing cancer. For the test, rats or mice are exposed to a chemical and then their blood is drawn and mixed with a fluorescent antibody to detect CD59. If the cells from the treated animals look dim to the flow cytometer compared to animals that were not exposed to a chemical, then it is inferred that the Pig-a gene has been mutated, and therefore inactivated, by the chemical.
The Pig-a gene mutation assay has been rapidly adopted by genetic toxicologists and is close to becoming an internationally accepted assay for identifying whether chemicals are mutagenic and, therefore, potentially carcinogenic. However, because the assay does not directly evaluate changes in the DNA sequence of the Pig-a gene, the question remains as to whether the phenotype (loss of CD59) really is a consequence of the genotype (a mutated Pig-a gene) and not due to some other biological process.
Because RBCs do not have a nucleus, Revollo et al. investigated whether erythroid cells, which are nucleated precursor cells that eventually give rise to RBCs during the process of hematopoiesis, acquire chemically-induced mutations in the Pig-a gene, resulting in loss of CD59. Unlike easily accessible RBCs, erythroid cells live in the bone marrow. Revollo et al. exposed rats to one of two chemicals, procarbazine or N-propyl-N-nitrosourea, which are known to cause mutations. Using a stringent DNA sequencing approach, they detected distinct mutational fingerprints of these chemicals in the Pig-a genes of erythroid cells that were also deficient in expression of CD59. There were no mutations in Pig-a in erythroid cells that expressed CD59. They also found that the fraction of RBCs that lacked CD59 reflected the fraction of erythroid cells with mutations in Pig-a. These findings provide strong evidence that the phenotype detected by the Pig-a assay reflects the genotype of Pig-a mutations caused by exposure to mutagenic chemicals, bringing the assay closer to wide-spread acceptance by regulatory agencies for the protection of public health.