Synonymous but Not Silent: A Synonymous VHL Variant in Exon 2 Confers Susceptibility to Familial Pheochromocytoma and von Hippel-Lindau Disease

Abstract

Context von Hippel-Lindau (VHL) disease, comprising renal cancer, hemangioblastoma, and/or pheochromocytoma (PHEO), is caused by missense or truncating variants of the VHL tumor-suppressor gene, which is involved in degradation of hypoxia-inducible factors (HIFs). However, the role of synonymous VHL variants in the disease is unclear. Objective We evaluated a synonymous VHL variant in patients with familial PHEO or VHL disease without a detectable pathogenic VHL mutation. Design We performed genetic and transcriptional analyses of leukocytes and/or tumors from affected and unaffected individuals and evaluated VHL splicing in existing cancer databases. Results We identified a synonymous VHL variant (c.414A>G, p.Pro138Pro) as the driver event in five independent individuals/families with PHEOs or VHL syndrome. This variant promotes exon 2 skipping and hence, abolishes expression of the full-length VHL transcript. Exon 2 spans the HIF-binding domain required for HIF degradation by VHL. Accordingly, PHEOs carrying this variant display HIF hyperactivation typical of VHL loss. Moreover, other exon 2 VHL variants from the The Cancer Genome Atlas pan-cancer datasets are biased toward expression of a VHL transcript that excludes this exon, supporting a broader impact of this spliced variant. Conclusion A recurrent synonymous VHL variant (c.414A>G, p.Pro138Pro) confers susceptibility to PHEO and VHL disease through splice disruption, leading to VHL dysfunction. This finding indicates that certain synonymous VHL variants may be clinically relevant and should be considered in genetic testing and surveillance settings. The observation that other coding VHL variants can exclude exon 2 suggests that dysregulated splicing may be an underappreciated mechanism in VHL-mediated tumorigenesis.

Case report: a synonymous VHL mutation (c.414A > G, p.Pro138Pro) causes pathogenic familial hemangioblastoma through dysregulated splicing

“…Using the fibroblasts derived from three patients and two healthy individuals, we confirmed that the c.414A > G mutation led to VHL exon 2 skipping and generated less E1E2E3 but more E1E3, consistent with previous reports [17,18]. Mechanistically, c.414A > G mutation may dysregulate the exonic splicing enhancer in exon 2 and cause exon 2 skipping [17].…”

“…The findings from this study and others [17,18], strongly advocate changing the status of VHL c.414A > G variant from "Uncertain significance" to "Pathogenic" for VHL disease in human variant databases (e.g. Clin-Var).…”

“…This mutation is a recurrent synonymous mutation in VHL disease. Based on this and two additional recent studies [17,18], the mutation has been reported in 29 individuals of 8 independent families (Supplementary Table S2). The overall association is with Type 2A (i.e.…”

“…diagnosis of PHEO and HGB but rarely RCC). Although this variant was shown to be associated with PHEO [17,18], the clinical information on its role in HGB was limited. In the clinic, HGB instead of RCC is the major contributor to the unfavorable overall survival of VHL patients [20], highlighting the importance of understanding the etiology of HGB.…”

“…E1E3 encodes a protein of 172 amino acids (pVHL 172 ) with generally low expression abundance and a possible lack of the tumor suppressor function due to the disruption of the HIF-binding domain [16]. Recent studies suggest that synonymous mutations of VHL can also lead to dysregulated splicing [17,18]. However, the clinical and molecular evidence to support the role of synonymous mutations in VHL disease is still very limited.…”