Functional study of synonymous mutations in VHL E2. (A) Functional hypoxia response element (HRE)–dependent reporter assays were performed in 786-O cells to evaluate the impact of VHL mutations in E2 (P138L and G144R) on VHL protein activity. The VHL protein lacking E2 (pVHL172/VHLΔE2) was used as a negative control. The results are expressed as firefly luciferase activity relative to Renilla luciferase as an internal control; 1.0 unit denotes the basal activity of endogenous HIF-2α using the HRE luciferase reporter plasmid. Immunoblots using an antibody specific for the hemagglutinin tag (HA) were used to detect HA-VHL. (B) Characterization of VHL E2 skipping by minigene analyses. Minigene experiments were performed in a variety of cell lines relevant to the studied diseases: renal (293T, 786-O, and HK2), pheochromocytoma (pheo; PC12), erythroid cell line (UT7 cultured with erythropoietin), and LCL. RT-PCR was performed on mRNA obtained from cell lines transfected with a minigene construct containing VHL E2 flanked by intronic sequences cloned between the SERPING1 exons (exons A and B; targeted by the RT-PCR primers). Bands corresponding to EA and EB spliced together or with VHL E2 are indicated on the right. (C) Sashimi plots from RNA-seq data. The positions of the different VHL exons are indicated, with the maximum number of reads for each exon indicated at the right. Splice junctions are denoted by the horizontal links, with details provided in supplemental Figure 15A. (D) Heatmap of pheochromocytoma transcriptome data. A comparison of transcriptome data for the pheochromocytoma from patient F11 III.1 (with P138P mutation) vs Affymetrix data from the largest available cohort of paragangliomas/pheochromocytomas (recruited by the French COMETE network) that identified homogeneous molecular subgroups associated with susceptibility genes (Burnichon et al³⁰ ).