Future directions
More research is required to assess the effectiveness of detecting genotoxic exposures in humans using the PIG-A assay, especially in different blood cell types (Bonetto et al., 2021). International collaborative projects are required and are anticipated to provide further evidence of the application of the human PIG-A assay. Studies are necessary to understand the role of this reporter gene in detecting genotoxin exposure in order to interpret future research findings. We also need to assess the long term follow up of patients following baseline PIG-A measurement to link the level of mutant frequency to development of later cancers and other chronic disease. This has proved to be fascinating in the lymphocyte MN field (Bonassi et al., 2007) and can be carried out using long-term epidemiological studies.
Moreover, it is not only crucial to determine the individual consequences of high levels of PIG-A mutant cells but also to confirm the degree to which GPI anchor deficiency is comparable toPIG-A mutation. Confirmation of PIG-A mutation has been carried out in GPI-deficient human T-lymphocytes (Ware et al., 2001) and granulocytes (Araten et al., 1999). Base-pair substitutions, small frameshift insertions and large deletions were identified in GPI-deficient T-lymphocytes isolated using aerolysin selection (Ware et al., 2001). PIG-A mutations including base-pair changes leading to single amino acid replacements were identified in granulocytes isolated by flow sorting from healthy volunteers. All mutations were found to either interfere with protein function or lead to protein truncation (Araten et al., 1999). Although confirmatory mutant sequencing is impossible in anucleate erythrocytes, periodic assessment in alternative blood cell types may provide information regarding the nature of mutation and also the type of genotoxic exposure.
Although there is still some uncertainty about whether erythrocytes are a suitable cell population for a human PIG-A assay, the proof-of-concept studies in the current literature indicates that the assay could be helpful for monitoring populations exposed to potential genotoxins. Possible clinical applications of the assay may include the diagnosis of DNA repair-deficient cancer-prone ’mutator’ phenotypes or monitoring chemotherapy patients for drug-induced mutation as a predictor of susceptibility to the formation of secondary tumours.
Another area for refinement for this test is the need for standard protocols for not only sample preparation but also data analysis. When measuring such rare cells, any subtle changes in the staining protocol or data acquisition, for example defining mutant gates on the flow cytometer can have an impact on results. Whilst the publishedPIG-A mutant data in healthy controls is remarkably consistent, a standard protocol would potentially minimise inter-laboratory variation and allow for comparison of results between research groups. Furthermore, the true potential of the PIG-A assay will be observed when we can overcome the issue of sample batching and are able to provide this test in low-income countries where mutagenic hazards and exposure-related diseases are often more common.
Combining this assay with others focussed on different mutational endpoints, such as the MN test or Comet assay, can enhance its effectiveness (Cao et al., 2023). Mutagenic exposures that act through different mechanisms and may give contrasting results in tests that measure a particular endpoint. A combination of tests including CBMN (Fenech et al., 2011), Comet (Milic et al., 2021) hypoxanthine-guanine phosphoribosyltransferase (HPRT) (Townsend et al., 2018) and\(\gamma\)H2AX assay (Kopp, Khoury, & Audebert, 2019) may improve test sensitivity and inform us about the nature of exposures. Research in our group indicates that the correlation between individual PIG-Amutant frequency and lymphocyte MN levels is not good with individuals often having high levels of PIG-A mutation or MN, but not vice versa (Figure 5). This suggests that using only one test is not sufficient to detect elevated levels of DNA damage in all individuals. Furthermore, when more than one DNA damage endpoint (PIG-A , micronuclei or COMET) has been measured in humans in response to a genotoxic exposure, positive associations have not been found with all endpoints. This could be due to the test used; for example, the RBCPIG-A assay may represent an accumulation of exposure over a prolonged period given the time taken for immature RBCs or haematopoietic stem cells to mature into PIG-A mutant RBCs. Given that the DNA damage evaluated in the COMET assay can be repaired, it is perhaps not surprising that not all genotoxins (i.e., lead) produce a positive response (Cao, Wang, Xi, et al., 2020). Aneugenic or clastogenic type DNA damage that can be observed in the lymphocyte micronucleus assay is essentially a snapshot of damage detected at the time of blood sampling which may otherwise be repaired in vivo so represents a shorter time window between exposure and measurable blood cell effects. This short time frame of opportunity may also be the case for the COMET assay. Together with the mechanistic differences of these endpoints and the differences in fate of the mutated cells, proves the need for the combination of complementary assays to comprehensively recognise genotoxic exposures (Torous et al., 2023).