Figure 4 Analysis of TLK2 Transcripts in Cell Lines
(A) Analysis of transcripts encoding nonsense mutations c.989C>A (p.Ser330∗) and c.2092C>T (p.Arg698∗) in cell lines of affected individuals. Left panel shows reverse transcriptase-PCR (RT-PCR) products of cDNA prepared from fibroblast and lymphoblastoid cell lines of subject with p.Ser330∗ variant, either in the presence (+C) or absence (−C) of cycloheximide and incubated with ApoI (digests wild-type allele). Central panel shows RT-PCR of cDNA prepared from lymphoblastoid cell line of subject with p.Arg698∗ variant, in the presence (+C) or absence (−C) of cycloheximide and incubated with Hpy99I (digests wild-type allele). Right panel shows proportion (±standard deviation) of variant alleles quantified by deep sequencing of triplicate samples. Statistical testing of differences: ∗p = 0.046; ∗∗p = 0.011; NS, not significant.
(B) Analysis of transcripts with canonical splice-site mutation c.1720+1G>T. A wild-type fragment at 300 bp in c.1720+1G>T lymphoblastoid cells is observed as well as a second fragment at 130 bp, which is absent in control cDNA. An increase of mutant transcript in cells was present when treated with cycloheximide (+C), indicating that the aberrant transcript was subject to NMD. Sequencing of the 300 bp (white box) and 130 bp (green box) fragments demonstrated skipping of exon 18 in the lower cDNA product. Abbreviations: Fibs, fibroblasts; EBV, lymphoblastoid cells; C/CHX, cycloheximide; WT, control cDNA.