Expression study of the new VHL transcript isoforms in patient cells. (A) Sashimi plots from RNA-seq data obtained from samples (LCLs or pheochromocytomas [Pheo]) of 3 patients from F8. The positions of the different VHL exons are indicated, with the maximal number of reads indicated at the right. Splice junctions are denoted by the horizontal links, with details provided in supplemental Figure 8. (B) TaqMan quantification of the different VHL isoforms from samples of F8 performed using probes specific to VHL E1-E2, the translated sequence upstream E1′, or E1-E1′ junctions. Relative gene expression was normalized to LCL control (C1) fixed at 1 (mean results of technical duplicates). (C) TaqMan quantification of the different VHL isoforms in LCLs (established from 2 independent controls and from patients of F1, F2, and F8) cultured in the absence or presence of puromycin (puro), an inhibitor of nonsense-mediated mRNA decay. TaqMan probes were specific to the VHL E1-E1′ or E1-E2 junction. The graph resumes experiments performed on LCLs cultured independently 3 times and quantified in duplicate. Data are represented as the mean ± standard error of the mean. (D) RT-PCR using primers specific for E1 and E3 was performed on mRNA extracted from LCLs established from controls and patients of F6 (carrying the mutation c.340+574A>T that targets the SA site of E1′). Patient with erythrocytosis is indicated in red. On the right, the spliced isoforms are schematically represented. **P < .05, ***P < .01 based on Student t test. *Denotes larger fragments that contained E1′ spliced with E1, but with 15 nucleotides deleted (represented in red) by the use of an alternative SA site (sequences of the cloned bands are detailed in supplemental Figure 10).