Fig. 4: COL25A1 transcript analysis in fibroblasts from subject 3 and a control.

Partial sequence electropherograms after direct Sanger sequencing of RT-PCR fragments obtained with a forward primer in exon 2 and a reverse primer in exon 4 (A) or a forward primer in exon 20 and a reverse primer in exon 24 (B) using cDNA from fibroblasts of a control (upper panel) and subject 3 (middle and lower panels). Subject 3’s fibroblasts were either untreated (middle panel) or treated with the NMD inhibitor cycloheximide (+CHX; lower panel) before RNA isolation. Exon numbering is given. Exon-exon junctions are marked by dotted lines. A Sequence traces show canonically spliced COL25A1 transcripts in control cells (upper panel). In untreated subject 3-derived fibroblasts only COL25A1 transcripts expressed from the maternal allele were identified as the reference guanine at position r.367 was detected (black arrow; middle panel). CHX treatment of subject 3 cells revealed transcripts with exon 2 directly spliced to exon 4 in COL25A1 transcripts (exon 3 skipping) in addition to canonically spliced transcripts (lower panel). Of note, the sequencing trace showing COL25A1 transcripts with exon 3 skipping (lower panel) are 70 bp shorter than those of canonically spliced COL25A1 transcripts, which explains why only peaks representing COL25A1 transcripts expressed from the maternal allele (with r.367g) are observed at the exon 3-exon 4 junction. B Partial sequence electropherograms show canonical splicing of exon 22 to exon 23 in control cells. In addition, a second sequence representing alternatively spliced COL25A1 transcripts with an exon 22-exon 24 junction is superimposed on the reference sequence (upper panel). In untreated and CHX-treated fibroblasts from subject 3, sequence traces of canonically spliced COL25A1 transcripts with the r.1198g > u variant (thymine marked by a red arrow) are observed in addition to alternatively spliced transcripts with skipping of exon 23 (superimposed on the reference sequence in middle and lower panels). alt. spl. alternatively spliced, ex exon, NMD nonsense-mediated mRNA decay.