Novel Compound VHL Heterozygosity (VHL T124A/L188V) Associated With Congenital Polycythaemia

Familial polycythaemia (erythrocytosis) is comprised of several inherited disorders arising from diverse aetiological factors. Primary polycythaemias are characterized by intrinsic defects within erythroid progenitors, which either grow independently or are hypersensitive to erythropoietin (EPO), while secondary polycythaemias are from circulating factors, such as EPO. Secondary familial polycythaemias are due to increased haemoglobin oxygen affinity (α, β, and γ globins and bisphosphoglycerate mutase mutations), while the primary familial polycythaemias are due to gain-of-function of EPO receptor (Gordeuk, et al 2005) and T-cell acute leukaemia 1 (TAL1) (Rogers, et al 2012) mutations. Mutations of the hypoxia-inducible factor (HIF) pathway present as either secondary (Tomasic, et al 2013) or with features of both primary and secondary polycythaemias (Chuvash polycythaemia) (Gordeuk, et al 2005). HIF pathway mutations include gain-of-function mutations of the EPAS1gene (also known as HIF2A) and loss-of-function mutations of two negative regulators of HIFs, EGLN1 (also known as PHD2) and von Hippel-Lindau (VHL) genes (Gordeuk, et al 2005). VHL, EGLN1 and EPAS1 mutations have been reported to be associated with either familial polycythaemia or pheochromocytoma/paraganglioma, but rarely both (Ladroue, et al 2008, Lorenzo, et al 2013). Two homozygous mutations of the VHL gene, Chuvash (VHL^R200W) and Croatian (VHL^H191D) polycythaemias, both in exon 3, have been reported to cause polycythaemia but not tumours (Ang, et al 2002, Tomasic, et al 2013).

We now report a 15-year-old male and his 10-year-old sister of mixed European descent, polycythaemic since infancy, with elevated EPO, who have been intermittently phlebotomized since their diagnosis. The parents are haematologically normal and not known to be related, with no history of VHL tumours among extended family members. Their peripheral blood was collected, with Institutional Review Board approved informed consents signed by both parents, and studied.

Mutational screening of the HIF pathway genes coding region at HIF1A, EPAS1 and EGLN1 were negative for any mutation. However, we found two missense mutations in the VHL genes from the two propositi. A novel heterozygous mutation in exon 2 NM_000551 c.370C>A (VHL: T124A) was found in the father and a previously reported pathogenic allele variant NM_000551 c.562C>G (VHL: L188V) (Ohh, et al 2000) in exon 3 in the mother; both propositi were compound heterozygotes for these changes.

Erythroid progenitors from the propositi and the father, but not the mother, were hypersensitive to EPO by peripheral blood burst-forming-unit-erythroid (BFU-E) colony assay (Fig. 1), a feature of primary polycythaemia (Ang, et al 2002, Gordeuk, et al 2005, Sokol, et al 1995). The relative expression of HIF target genes, HK1, SLC2A1 and TFRC, from three biological replicate samples was significantly increased when compared to controls (data not shown).

Figure 1. Erythroid progenitor sensitivity assay of erythropoietin (EPO).

Burst-forming-unit-erythroid (BFU-E) colonies were enumerated and expressed as a percentage of maximum at stimulation with different EPO concentrations from each family member and controls.

We further investigated the downstream effects of protein expression and function on E3 ligase activity in HIF regulation by expressing recombinant wild-type and mutant VHL protein constructs in a VHL-deficient 786-0 renal cell adenocarcinoma cell line and assessed their interaction with HIF1A protein by Western blot. Both wild-type and mutant VHL bound HIF1A protein comparably (Fig. 2A). We then tested ubiquitination of the HIF1A protein and HIF1 bound and ubiquitinated by both tested VHL mutants as efficiently as wild type VHL (Fig. 2B). These results suggest that the intrinsic biological function of these two VHL mutants is intact. We then considered the possibility that these two mutants could have decreased stability. To test this, we conducted a cycloheximide assay to study protein folding, maturation, and degradation. The results showed that the stability of both VHL¹²⁴ and VHL¹⁸⁸ mutants were significantly reduced, with their half-lives decreased from ~8 h to ~2 h (Fig 2C,D).

Figure 2. VHL structural and functional studies.

A–B: The efficient reduction of HIF1A by reintroduction of the VHL mutants into VHL-deficient 786-0 cells in contrast with wild-type and empty vector. C-D: Cyclohexamide (CHX) chase assay for protein half-life shows that mutant VHL proteins have decreased half-lives. 786-0 cells stably transfected with VHL variants and treated with CHX (200μM) for 4 h; hourly lysates were analysed by Western blot.

We then generated a crystal structure model of HIF-VHL-ElonginB-ElonginC Complex. The predicted protein structure indicated that both amino acid residues lie in the interior pocket of the VHL protein and are less likely to involve physical contact with HIF (data not shown).

In summary, we describe a novel germline VHL exon 2 mutation present in the compound heterozygous state with an exon 3 VHL variant in two polycythaemic siblings. We showed that this compound heterozygosity of VHL is associated with elevated EPO (secondary polycythaemia) and also has characteristics of primary polycythaemia (EPO hypersensitivity). In addition, the heterozygous VHL^T124A mutation present in the father also exhibited BFU-E hypersensitivity. We then evaluated HIF activity by transcription of HIF-target genes. Granulocytes from compound VHL^{T124A, L188V} subjects had increased transcripts of the HIF-regulated genes SLC2A1 and TFRC, which correlated with delayed HIF degradation due to a reduced half-life of VHL. It has been reported that VHL mutations accompanied by BFU-E EPO hypersensitivity are unique for distal mutations in VHL exon 3 (VHL^H191D and VHL^R200W) and are due to altered SOCS1 binding (a negative regulator of JAK2)(Russell, et al 2011). However, we also demonstrated BFU-E EPO hypersensitivity in these proximal exon 2 VHL mutations (Lanikova, et al 2013), indicating that other, yet to be defined molecular mechanism(s) of EPO-hypersensitivity must exist. We showed that the loss of function of VHL mutations in this family with congenital polycythaemia is, at least in part, due to decreased stability of mutant VHL proteins resulting in delayed ubiquitinization and degradation of HIF, and this increases HIF1A activity.