F12c.414A>G/WTVHL p.Pro138Pro/WTNNNNVHL disease—

Figure 6. 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).

unilateral PHEOs (Fig. 1a): her father (II:1) at age 73, her 34-year-old brother (III:2), her paternal aunt in her 40s (II:4), and the aunt’s 12-year-old son (III:7)

Figure 1.

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c.414A>G, p.Pro138Pro

The p.(Pro138Pro) variant was identified in a patient with a family history of VHL disease and was functionally validated by mRNA analysis, which showed a splicing alteration (data not shown)

F12c.414A>G/WTVHL p.Pro138Pro/WTNNNNVHL disease—

Expression study of the new VHL isoforms in patients' cellsRNA sequencing (RNA-seq) analysis of patients' LCLs and tumors demonstrated that genomic variants in E1′ were associated with an upregulation of transcripts containing the E1′ cryptic exon compared with controls (Figure 3A; supplemental Figure 8). In addition, the mutated allele was preferentially expressed (>70%), suggesting a causal relationship between the genetic variants and E1′ retention (supplemental Figure 8C).Figure 3. 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).Download : Download high-res image (230KB)Download : Download full-size imageFigure 3. 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).We performed quantitative RT-PCR using TaqMan probes specific for the different splicing junctions or for the region upstream of E1′ (location of probes shown in Figure 1C), which showed a strong upregulation of a unique isoform containing E1′ spliced with E1 in LCLs and tumors from patients of F8 compared with controls (Figure 3B). The study therefore focused on the isoform E1-E1′. Transcripts that retained E1′ spliced with E1 contained (in-frame with E1) a premature termination codon (Figure 1C) and could likely be targeted for degradation according to nonsense-mediated mRNA decay (NMD) mechanisms. We therefore investigated the expression of transcripts that contained E1 spliced with E1′ in the absence or presence of puromycin, an inhibitor of NMD. Without puromycin, higher levels of expression were seen in samples with mutated E1′ (Figure 3C) compared with samples containing WT E1′. Exposure to puromycin resulted in a profound induction of isoforms containing E1 spliced with E1′ vs other isoforms (Figure 3C; supplemental Figure 9). These results indicated that isoforms with E1-E1′ junction were degraded by NMD and may have failed to produce proteins. We performed RT-PCR on LCL samples of F6 carrying the mutation in the SA site, using primers in VHL exons E1 and E3. We observed fragments of larger size in samples from patients carrying the mutation, which were highly visible in the presence of puromycin (Figure 3D). Cloning and sequencing of these fragments demonstrated a dysregulated splicing of E1′ with the use of an alternative SA site located 15 nucleotides downstream (supplemental Figure 10).To measure the impact of the splicing dysregulation on pVHL expression, we performed an immunoblot with an antibody able to recognize the amino acids encoded by E1 and therefore able to detect the different pVHL isoforms.8 The antibody detected overexpressed exogenous X1 protein after transfection of the plasmid encoding X1 (Figure 4A). However, it failed to detect the endogenous protein, even in patients' samples that overexpressed the mRNA containing E1-E1′. Instead, the immunoblots showed a lower expression of all the VHL protein isoforms in patients with mutated E1′ (Figures 4A-B).Figure 4. Functional studies of mutations in VHL E1′. (A-B) Immunoblot analysis of patient LCLs. A representative immunoblot (A) and quantification of 3 different immunoblots (B) are presented. Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, and results obtained with LCL control (C1) were fixed at 100%. Data are represented as the mean ± standard error of the mean. A VHL antibody that recognized VHL E1 was able to detect the hypothetical X1 protein. In the most left lane, control LCLs were transfected with an expression vector containing the coding sequence for X1; 5 µg of proteins were blotted vs 45 µg for other samples to avoid signal saturation with overexpressed X1. Patients with erythrocytosis are indicated in red. (C) Functional hypoxia response element (HRE)–dependent reporter assays were performed in 786-O cells (ie, VHL− cells that constitutively express HIF-2α). The results are expressed as relative firefly luciferase activity with Renilla luciferase as an internal control; 1.0 unit denotes the basal activity of endogenous HIF-2α using the HRE luciferase reporter plasmid. The ability of WT and mutated X1 to downregulate firefly luciferase activity (related to HIF activity) was compared with pVHL and in competition with pVHL. An immunoblot using an antibody specific to the hemagglutinin tag (HA) was used to detect HA-VHL and HA-X1. X1-L128V+L138P corresponds to a potential impact of the c.340+648T>C and c.340+617C>G mutations on the hypothetical X1 protein. Three independent experiments were performed. (D) Characterization of VHL E1′ retention by the minigene experiment (representative picture of agarose gel; n = 3). RT-PCR was performed on mRNA obtained from cell lines transfected with a minigene construct containing VHL E1′ (WT or mutated) flanked by large intronic sequences cloned between the SERPING1 exons (exons A and B, targeted by the RT-PCR primers). The plasmids were transfected, and the expression of the spliced chimeric transcripts (containing EA and EB ± E1′) was analyzed. Two WT constructs containing E1′ were used; 1 contained the single-nucleotide polymorphism c.340+1150T>C (rs779808). Bands corresponding to EA and EB spliced together or with VHL E1′ are indicated on the right. *P < .05, **P < .01, ***P < .001, ****P < .0001 based on Student t test. †Corresponds to unspecific bands verified by sequencing. The minigene experiment performed with the construction carrying the mutation c.340+574A>T (that targets the SA site of E1′ in F6) confirmed the use of an alternative SA site (right panel) with the deletion of 15 nucleotides (represented in red). ns, not significant.Download : Download high-res image (204KB)Download : Download full-size imageFigure 4. Functional studies of mutations in VHL E1′. (A-B) Immunoblot analysis of patient LCLs. A representative immunoblot (A) and quantification of 3 different immunoblots (B) are presented. Relative gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, and results obtained with LCL control (C1) were fixed at 100%. Data are represented as the mean ± standard error of the mean. A VHL antibody that recognized VHL E1 was able to detect the hypothetical X1 protein. In the most left lane, control LCLs were transfected with an expression vector containing the coding sequence for X1; 5 µg of proteins were blotted vs 45 µg for other samples to avoid signal saturation with overexpressed X1. Patients with erythrocytosis are indicated in red. (C) Functional hypoxia response element (HRE)–dependent reporter assays were performed in 786-O cells (ie, VHL− cells that constitutively express HIF-2α). The results are expressed as relative firefly luciferase activity with Renilla luciferase as an internal control; 1.0 unit denotes the basal activity of endogenous HIF-2α using the HRE luciferase reporter plasmid. The ability of WT and mutated X1 to downregulate firefly luciferase activity (related to HIF activity) was compared with pVHL and in competition with pVHL. An immunoblot using an antibody specific to the hemagglutinin tag (HA) was used to detect HA-VHL and HA-X1. X1-L128V+L138P corresponds to a potential impact of the c.340+648T>C and c.340+617C>G mutations on the hypothetical X1 protein. Three independent experiments were performed. (D) Characterization of VHL E1′ retention by the minigene experiment (representative picture of agarose gel; n = 3). RT-PCR was performed on mRNA obtained from cell lines transfected with a minigene construct containing VHL E1′ (WT or mutated) flanked by large intronic sequences cloned between the SERPING1 exons (exons A and B, targeted by the RT-PCR primers). The plasmids were transfected, and the expression of the spliced chimeric transcripts (containing EA and EB ± E1′) was analyzed. Two WT constructs containing E1′ were used; 1 contained the single-nucleotide polymorphism c.340+1150T>C (rs779808). Bands corresponding to EA and EB spliced together or with VHL E1′ are indicated on the right. *P < .05, **P < .01, ***P < .001, ****P < .0001 based on Student t test. †Corresponds to unspecific bands verified by sequencing. The minigene experiment performed with the construction carrying the mutation c.340+574A>T (that targets the SA site of E1′ in F6) confirmed the use of an alternative SA site (right panel) with the deletion of 15 nucleotides (represented in red). ns, not significant.Functional characterization of the mutated VHL E1′Because the hypoxia pathway represents the major pathway involved in the genesis of secondary erythrocytosis and VHL disease,13, 14, 1528 we performed functional studies of the hypothetical WT or mutated X1 proteins using hypoxia response element–dependent reporter assays. These functional tests failed to reveal any substantial effects of X1 on this pathway, either alone or in competition with pVHL (Figure 4C; supplemental Figure 11). We next focused our study on a potential impact of the E1′ variants on splicing by performing splicing reporter minigene assays in various cell lines. The experiments showed that splicing of WT E1′ was barely detected (Figure 4D). Interestingly, higher molecular weight bands corresponding to the expected spliced isoforms containing E1′ appeared in the presence of the mutations. Cloning and sequencing of the isoforms confirmed a retention of E1′ at the expected splicing sites. The level of expression of this upper isoform, specific to E1′ inclusion, was higher for mutations associated with cancers than for those with erythrocytosis, independently of the cell lines. Notably, the combination of both variants associated with cancer had a more pronounced effect than each variant individually (the single-nucleotide polymorphism showing very low effect), suggesting a synergistic effect of the variants on splicing (supplemental Figure 12). In silico analysis of c.340+617C>G consistently predicted a severe alteration of splicing by the creation of an exonic splicing enhancer site (supplemental Figure 13).The minigene experiment performed with mutated E1′ at the SA site (F6) confirmed the inclusion of E1′ during splicing (Figure 4D, right panel), using the same downstream alternative SA site identified in mRNA extracted from patients' LCLs (supplemental Figure 10).Synonymous mutations in VHL E2 induce exon skippingWe then focused our study on the synonymous heterozygous D143D mutation in E2, identified in the proband of F1. This mutation was also identified in the homozygous state in 2 patients with erythrocytosis (F9 and F10; Figure 5A; Table 1). The mRNA extracted from LCLs established from the different members of these 2 families was reverse transcribed and sequenced. Comparison of chromatograms obtained by sequencing of DNA vs cDNA displayed a weaker peak of the mutated allele in cDNA, reflecting a decreased expression of the mRNA transcripts carrying the mutation.Download : Download high-res image (221KB)Download : Download full-size imageFigure 5. 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.A different synonymous mutation in E2, c.414A>G p.Pro138Pro (P138P), was identified in 2 families (F11 and F12) with VHL disease (Figure 5B). This heterozygous mutation segregated with the disease in 3 generations. Sequencing of DNA extracted from the pheochromocytoma of F11-III.1 showed a loss of the WT allele in the tumor, demonstrating loss of heterozygosity as currently described in classical VHL disease.Suspecting an effect of the synonymous mutations on splicing, we next assessed the expression of the VHL transcripts in patients' samples by RT-PCR (Figure 5C). We found a significant change in the ratio of expressed VHL isoforms, with a higher expression of the E1-E3 transcripts in LCLs of patients homozygous for D143D and in the pheochromocytoma with P138P mutation. These results suggest an effect of the mutation on splicing regulatory elements, leading to E2 skipping. In silico analysis of the synonymous mutations indicated a potential effect on exonic splicing enhancer motifs (supplemental Figure 14).To specifically quantify the different VHL isoforms, we performed quantitative RT-PCR on LCLs using TaqMan probes complementary to the VHL E1-E2 or E1-E3 junction (Figure 5D). All mutated samples displayed an increase of the isoform with skipped E2 (E1-E3). The patients carrying a homozygous D143D mutation presented a severe decrease of the WT isoform expression level. These results demonstrated that synonymous mutations in E2 affected mRNA isoform production.To evaluate a potential impact on protein expression, we performed a western blot analysis. We did not observe any overexpression of the pVHL172 isoform as expected from mRNA quantification studies. However, in patients homozygous for D143D, we detected a strong downregulation of all the pVHL isoforms, which was also observed to a lesser extent in LCL samples from heterozygous patients (Figure 5E).Proportion of VHL E2 skipping is correlated with disease severity in minigene experimentsTo study the impact of the synonymous E2 mutations on splicing in different cell lines, we performed minigene assays. We tested the implications of the D143D and P138P mutations on splicing in addition to the nearby mutations described in patients with erythrocytosis (P138L and G144R).4,6,29 Indeed, these mutations may also affect splicing rather than VHL protein function. We first evaluated the loss of function of these mutants by reporter assays. We found that these could downregulate HIF in a manner similar to the WT protein, in contrast to VHL proteins lacking E2 (pVHLΔE2), which are described as nonfunctional in terms of their regulation of HIF activity8, 9, 1012 (Figure 6A). Minigene experiments were performed in a variety of cell lines relevant to the studied diseases, using LCLs as control (Figure 6B). We demonstrated that all mutations caused E2 skipping in LCLs. In other cell lines, these experiments demonstrated a major impact on the VHL E2 splicing of the P138P mutation, which is associated with cancer. The mutations P138L, D143D, and G144R, which are all associated with erythrocytosis, displayed a weaker effect on splicing, with slight variations among cell lines but with a stronger effect in the erythroid cell line (UT7 cultured with erythropoietin).Figure 6. 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).Download : Download high-res image (254KB)Download : Download full-size imageFigure 6. 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).Splicing dysregulation of VHL is causal in the development of diseaseRNA-seq confirmed that the synonymous mutations were not silent but instead induced potent E2 skipping (Figure 6C; supplemental Figure 15A). Additional transcriptome analyses of the pheochromocytoma carrying P138P showed an upregulation of HIF target genes (typically seen in VHL-related pheochromocytomas) compared with the pheochromocytoma carrying a RET mutation (supplemental Figure 15B).30 We then reanalyzed our independent cohort of pheochromocytomas30 using RNA-seq data from both tumors. After unsupervised classification, we observed a segregation of the P138P pheochromocytoma with other VHL-related tumors (C1B cluster), whereas the control pheochromocytoma bearing the RET mutation was grouped with other RET-related tumors (Figure 6D).

87VHLE2c.414A>Gp.P138PPP30946460M363622NoPCC2.3No

VHLE2c.414A>Gp.P138P

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unilateral PHEOs (Fig. 1a): her father (II:1) at age 73, her 34-year-old brother (III:2)

Figure 1.