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Both PIGA and PIGL mutations cause GPI ‐a deficient isolates in the T k6 cell line
Author(s) -
Nicklas Janice A.,
Carter Elizabeth W.,
Albertini Richard J.
Publication year - 2015
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.21953
Subject(s) - biology , gene , microbiology and biotechnology , mutation , point mutation , genetics , intron , mutant , allele , cell culture , breakpoint , chromosomal translocation
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. © 2015 Wiley Periodicals, Inc.