Fig. 3: Verification of the effect of the c.16-2063 A > C variant located in intron 1 in Patient 3 by reverse transcription PCR (RT-PCR) of exons 1–3 of HGD.
From: A novel deep intronic variant strongly associates with Alkaptonuria
a Agarose gel electrophoresis analysis reveals, other than the normal product (arrow), several larger-than-expected fragments in the patient (star). b Real-time PCR analysis reveals that the total amount of exons 1–3 HGD RNA was higher in Patient 3 than in the control. c High-resolution melting analysis reveals a shift in melting temperature in the patient. d NGS analysis reveals the inclusion of a 126-bp cryptic exon (283 reads) in half of the products (446 reads for exon 2) in the patient. A small portion of the reads in the control also contains this cryptic exon. There are other less-frequent cryptic exons included in the patient (not shown). e A zoom-in view of the cryptic exon reveals a major transcript (arrow) that causes protein truncation (Tyr5_Ile6insValTer17), and a minor transcript that also causes protein truncation (open arrow). f The c.16-2063 A > C variant is predicted to disrupt an SRSF5-binding site (TATCAGG) and then activate the inclusion of the cryptic exon (genomic coordinate 3:120,396,751–120,396,876).
