Both PIGA and PIGL mutations cause GPI‐a deficient isolates in the Tk6 cell line

Abstract

Molecular analysis of proaerolysin selected glycosylphosphatidylinositol anchor (GPI-a) deficient isolates in the TK6 cell line was performed. Initial studies found that the expected X-linked PIGA mutations were rare among the spontaneous isolates but did increase modestly after ethyl methane sulfate (EMS) treatment (but to only 50% of isolates). To determine the molecular bases of the remaining GPI-a deficient isolates, real-time analysis for all the 25 autosomal GPI-a pathway genes was performed on the isolates without PIGA mutations, determining that PIGL mRNA was absent for many. Further analysis determined these isolates had several different homozygous deletions of the 5’ region of PIGL (17p12-p22) extending 5’ (telomeric) through NCOR1 and some into the TTC19 gene (total deletion >250,000bp). It was determined that the TK6 parent had a hemizygous deletion in 17p12-p22 (275,712bp) extending from PIGL intron 2 into TTC19 intron 7. Second hit deletions in the other allele in the GPI-a deficient isolates led to the detected homozygous deletions. Several of the deletion breakpoints including the original first hit deletion were sequenced. As strong support for TK6 having a deletion, a number of the isolates without PIGA mutations nor homozygous PIGL deletions had point mutations in the PIGL gene. These studies show that the GPI-a mutation studies using TK6 cell line could be a valuable assay detecting point and deletion mutations in two genes simultaneously.

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

“…In terms of deletion frequency at PIGA versus HPRT1 , HPRT clonal assays were also performed on the same set of Veterans allowing for comparison of mutation spectra between the two genes. While PIGA and HPRT1 have similar percentages of point mutations, frameshifts, insertions, splice, and complex mutations, PIGA had twice as many frameshifts and small deletions but less than one‐third as many large deletions as compared to HPRT1 (Nicklas et al, , ). This dearth of deletions at PIGA suggests PIGA has much closer vital genes than HPRT1 which both limit deletion size and deletion frequency.…”

“…At HPRT1 , exon 2–3 deletions are often the result of V(D)J recombinase‐mediated events especially in children (Fuscoe et al, , ; Finette et al, ). For example, 11 of the 1,050 HPRT1 mutants from these Veterans were V(D)J recombinase‐mediated deletions (Nicklas et al, ). However, there was no indication that any of the PIGA deletions found here were V(D)J recombinase‐mediated.…”

“…Real-time analysis was performed as described in Nicklas et al (2015b). Samples were run with the control beta-actin (ACTB) on each plate.…”

Suitability of Long‐Term Frozen Rat Blood Samples for the Interrogation of Pig‐a Gene Mutation by Flow Cytometry

“…For instance, in the case of rat erythrocytes, fluorochrome‐conjugated antibodies against the GPI‐anchored antigen CD59, together with flow cytometric analysis, are used to score the incidence of nonfluorescent mutant cells relative to fluorescent wild‐type cells. DNA‐sequencing results clearly support the use of the GPI anchor‐deficient phenotype as a reliable reporter of Pig‐a gene mutation (Kimoto et al, ; Dobrovolsky et al, ; Nicklas et al, ; Revollo et al, , ; Krüger et al, ; Bemis et al, ).…”

The Pig‐a Gene Mutation Assay in Mice and Human Cells: A Review

“…The induced mutation spectra obtained were consistent with expected spectra obtained in other endogenously expressed genes, verifying the validity of the mutation frequencies measured. In human cells, point mutations as well as deletions in PIG‐A have been identified in TK6 cell lines . Recently, heterogeneous pools of Pig‐a ‐mutant T cells derived from DMBA‐exposed rats were efficiently sequenced by an elegant novel technique named mutation analysis with random DNA identifiers (MARDI), requiring no clone‐by‐clone analyses .…”