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).