Background: Somatic variants in endothelial PAS domain-containing protein 1 (EPAS1), encoding hypoxia-inducible transcription factor 2-alpha (HIF2 have been reported in patients with polycythaemia and multiple paragangliomas (PGL). Our genetic testing of heritable phaeochromocytoma (PC) and PGL identified a number of germline EPAS1 variants in subjects who also carrying pathogenic VHL or SDHB mutations. EPAS1 variants were also identified in some PC/PGL patients without an identified germline mutation in any other known PC/PGL susceptibility gene.
Objective: Assess the functional consequences of germline EPAS1 variants in PC/PGL.
Method: Six germline EPAS1 variants were identified in nine PC/PGL patients: p.His194Arg, p.Arg247Ser, p.Phe374Tyr, p.Thr766Pro, p.Pro785Thr and p.Ile789Val. In vitro studies assessed stability of mutant HIF2 under normoxia, and effects on interaction with regulatory (VHL) and response (ARNT) proteins. GFP-tagged HIF2 expressing mutant and wild-type constructs (including p.Pro531Thr, as a positive control) were transfected into HEK293 cells. Stability of HIF2a and effects on interacting partners were assessed by Western blot and co-immunoprecipitation (Co-IP) assays, while effects on transcriptional response and downstream target genes (CCND1 and SLC2a) were assessed by luciferase reporter assay and qRT PCR.
Results: HIF2 mutants p.Arg247Ser, p.Phe374Tyr, p.Pro531Thr and p.Pro785Thr were more stable than wild-type, under normoxia. Further, significantly reduced interaction was observed between VHL and HIF2 p.Arg247Ser and p.Pro531Thr constructs (p<0.05), while no effects were observed between ARNT and any of the HIF2 mutants. HIF2 variants p.Phe374Tyr, p.Pro531Thr and p.Pro785Thr stimulated transcription of target genes (p<0.05), while p.Pro531Thr was the only variant to induce expression of both CCND1 and SLC2A (p<0.05).
Conclusion: Germline EPAS1 variants p.Arg247Ser and p.Phe374Tyr share some functional features in common with the known oncogenic somatic variant p.Pro531Thr. Although germline EPAS1 variants appear to have modest effects on interacting partners and downstream targets, they may be acting as modifiers of PC/PGL.