Genetic and expression study of synonymous mutations in VHL E2. (A-B) Pedigree and sequence chromatograms of germline DNA, tumor (pheochromocytoma) DNA, or cDNA prepared from 2 families (F9 and F10) with erythrocytosis (A) and 2 families (F11 and F12) with VHL disease (B). (C) Results of RT-PCR using mRNA extracted from LCLs (F9 and F10) and leukocytes and tumor material (pheochromocytoma) (F11 and F12). (D) TaqMan quantification of the different VHL isoforms in LCLs (established from patients of F9, F10, and F11) cultured in the absence or presence of puromycin. TaqMan probes are specific to the VHL E1-E2 or E1-E3 junction. Relative gene expression was normalized to LCL control (C1). (E) Immunoblot analysis of patient LCLs. Patients with erythrocytosis are indicated in red. A representative immunoblot (upper panel) and quantification of 4 different immunoblots are displayed (lower panel). Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, and results obtained with C1 were fixed at 100%. Data are represented as the mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001, ****P < .0001 based on Student t test. CNS, central nervous system.