Molecular analysis of glycosylphosphatidylinositol anchor deficient aerolysin resistant isolates in gulf war i veterans exposed to depleted uranium

Abstract

During the First Gulf War (1991) over 100 servicemen sustained depleted uranium (DU) exposure through wound contamination, inhalation, and shrapnel. The Department of Veterans Affairs has a surveillance program for these Veterans which has included genotoxicity assays. The frequencies of glycosylphosphatidylinositol anchor (GPIa) negative (aerolysin resistant) cells determined by cloning assays for these Veterans are reported in Albertini RJ et al. (2019: Environ Mol Mutagen). Molecular analyses of the GPIa biosynthesis class A (PIGA) gene was performed on 862 aerolysin‐resistant T‐lymphocyte recovered isolates. The frequencies of different types of PIGA mutations were compared between high and low DU exposure groups. Additional molecular studies were performed on mutants that produced no PIGA mRNA or with deletions of all or part of the PIGA gene to determine deletion size and breakpoint sequence. One mutant appeared to be the result of a chromothriptic event. A significant percentage (>30%) of the aerolysin resistant isolates, which varied by sample year and Veteran, had wild‐type PIGA cDNA (no mutation). As described in Albertini RJ et al. (2019: Environ Mol Mutagen), TCR gene rearrangement analysis of these isolates indicated most arose from multiple T‐cell progenitors (hence the inability to find a mutation). It is likely that these isolates were the result of failure of complete selection against nonmutant cells in the cloning assays. Real‐time studies of GPIa resistant isolates with no PIGA mutation but with a single TCR gene rearrangement found one clone with a PIGV deletion and several others with decreased levels of GPIa pathway gene mRNAs implying mutation in other GPIa pathway genes. Environ. Mol. Mutagen. 60:470–493, 2019. © 2019 Wiley Periodicals, Inc.

Longitudinal study of t‐cell somatic mutations conferring glycosylphosphatidylinositol‐anchor deficiency in gulf war I veterans exposed to depleted uranium

“…As detailed in Nicklas et al (), in vivo clonality as determined by TCR gene rearrangement patterns was frequently observed in our molecular studies. Although we used analogous data to correct MFs for mutation frequencies (MutFs) in our HPRT studies, we have not done so with the current data set partly because post‐thymic clonality was much rarer among the PIGA mutants than the HPRT mutants (10 clones in 610 mutants vs. 62 clones in 1234 mutants).…”

“…Our analysis of the TCR gene rearrangement patterns among isolates with identifiable PIGA mutations also revealed multiple isolates from the same Veteran with the identical change in PIGA but with different TCR gene rearrangements (Nicklas et al, 2019). This pattern was observed more frequently than in our studies of HPRT mutations in these Veterans (7 observations in 610 PIGA mutants vs. 7 observations in 1234 HPRT mutants) (Albertini et al, 2015).…”

“…We describe here what we deem as failure of aerolysin selection to completely eliminate GPIa bearing protein cells in some of the cloning assays. These failures were recognized only after we had completed the molecular analyses described in Nicklas et al (). Incomplete selection by aerolysin in cloning assays for GPIa deficiency could possibly have been remedied by adding additional aerolysin midway through the culture interval.…”

“…These failures were recognized only after we had completed the molecular analyses Environmental and Molecular Mutagenesis. DOI 10.1002/em described in Nicklas et al (2019). Incomplete selection by aerolysin in cloning assays for GPIa deficiency could possibly have been remedied by adding additional aerolysin midway through the culture interval.…”

“…Nonetheless, we present them in detail for two reasons. First, mutant colonies isolated in these quantitative studies allowed molecular analyses and an initial description of a molecular mutational spectrum for background PIGA mutations arising in vivo in T‐lymphocytes in humans (Nicklas et al, ). Second, in the course of our molecular analyses, which of necessity followed the quantitative studies, we discovered that some cloning assays with aerolysin selection allowed outgrowth of pseudo‐resistant (nonmutant) cells, thereby inflating estimated MFs.…”