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

摘要

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 >250,000 bp). It was determined that the TK6 parent had a hemizygous deletion in 17p12-p22 (275,712 bp) 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. Environ. Mol. Mutagen. 56:663-673, 2015. (c) 2015 Wiley Periodicals, Inc.