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 >250,000 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.

Keywords: GPI-a; PIGA; PIGL; TK6; glycosylphosphatidylinositol anchor; in vitro; mutation.

Fig 1

PIGL genomic multiplex in two amplifications. TK6 is the parental cell line, BE has no PIGA or PIGL mutation, 50 and 63 both have the PIGL exon 1/exon 2 deletion. Lane 5 is the blank negative control.

Fig. 2

Real-time analysis of PIGL cDNA (A) and actin (ACTB) (B) positive control for amplification. CC, CX and DA have the PIGL type A deletion. TK6 is the parental line. DB and DC do not make PIGL mRNA for unknown reasons. CJ and CW have point mutations in PIGA.

Fig. 2

Real-time analysis of PIGL cDNA (A) and actin (ACTB) (B) positive control for amplification. CC, CX and DA have the PIGL type A deletion. TK6 is the parental line. DB and DC do not make PIGL mRNA for unknown reasons. CJ and CW have point mutations in PIGA.

Fig. 3

Extent of the homozygous PIGL deletions on chromosome 17p. Numbering is based on Genbank GRCh38 Primary Assembly annotation 106. Adapted from the NCBI “Genomic regions, transcripts, and products” diagram for PIGL (http://www.ncbi.nlm.nih.gov/gene/9487).

Fig. 4

Cytogenetic Analysis of the TK6 cell line. A. Karyotype, B. Closeups of chromosome 17, C. Fish results with a Smith-Magenis probe.

Fig. 4

Cytogenetic Analysis of the TK6 cell line. A. Karyotype, B. Closeups of chromosome 17, C. Fish results with a Smith-Magenis probe.

Fig. 4

Cytogenetic Analysis of the TK6 cell line. A. Karyotype, B. Closeups of chromosome 17, C. Fish results with a Smith-Magenis probe.

Fig. 5

Frequency of PIGA mutations, PIGL deletions and PIGL point mutations at different days of selection for EMS-treated TK6 cells.