Clinical and functional properties of novel VHL mutation (X214L) consistent with Type 2A phenotype and low risk of renal cell carcinoma

Abstract

This report describes clinical characteristics in families with a Type 2A phenotype and functional properties of a novel von Hippel Lindau variant (X214L). Pedigrees were analyzed. Analysis of von Hippel Lindau (VHL) coding exons and flanking intronic sequences in DNA from a proband with pheochromocytoma and islet cell tumor was performed. Western blot assays for VHL protein (pVHL), HIFα, and Jun B were conducted using VHL null renal clear carcinoma cell lines that were engineered to produce wild-type or X214L mutant pVHL. Pedigree analysis indicated that the variant tracked with disease and the same or similar VHL point mutations were identified in several Type 2A families. The predicted 14 amino acid extended pVHL variant, when reintroduced into VHL null cells, was stable and retained the ability to downregulate HIFα in a hydroxylationdependent manner. In contrast, the variant was defective with respect to downregulation of JunB. pVHL X214L, like other pVHL variants associated with a low risk of clear cell renal carcinoma, largely preserves the ability to downregulate HIF. In contrast, this variant, like other pVHL variants linked to Type 2A disease, fails to suppress JunB. This underscores that JunB may play a role in the pathogenesis of Type 2A VHL disease.

Fig 1

(a) DNA sequence analysis of the mutant VHL gene. The normal stop codon (TGA) is changed to a codon for leucine (TTA) at amino acid position 214, leading to an extension peptide containing 14 novel amino acids and a new stop codon. The original stop codon and the stop codon of the extension peptide are boxed. K, heterozygous for a G and a T at the same position in the DNA sequence. A portion of the translated amino acid sequence indicating the normal stop codon and the sequence of the extended peptide is shown below the nucleotide sequence. (b) Illustration of the 213 amino acids comprising the full-length VHL protein (pVHL). pVHL contains alpha (α) and beta (β) subdomains. The α subdomain binds to elongin C (C), Cullin 2 (Cul2), elongin B (B), and Rbx1. The beta subdomain is important for recognition and binding to substrates such as hypoxia-inducible factor 1 alpha. (modified from Ref (9)).

Fig 2

Pedigree of family with VHL X214L germline mutation. The index patient (proband) is indicated by . Family members with confirmed VHL gene mutations are identified by the plus sign (+). The square symbols represent males; the circle symbols represent females. The age (years) of each individual is noted below the symbol. Family members affected by cancer are identified by diagnoses abbreviations and symbols [pheochromocytoma (Pheo ), liver cancer (L ), cancer not otherwise specified (Ca–Nos ), Hodgkin's (H ), islet cell tumor of the pancreas (Islet), retinal hemangioblastoma (RHE )]. Diagonally dashed symbols represent deceased individuals.

Fig 3

Immunoblot analysis using two VHL protein (pVHL) null renal carcinoma cell lines (786-O and RCC-4). Where indicated, cells were treated with 1mM of dimethyloxaloylglycine (DMOG), dissolved in dimethyl sulfoxide (DMSO), for 16 h to induce HIF2α protein. DMOG-induced HIF2α protein in the X214L mutant (X) and wt VHL (V) expressing cells. Compared to wt VHL, high-levels of JunB expression was seen in the X214L mutant expressing cell lines, pre- and post-DMOG exposure. No pre- and post-DMOG expression differences were detected in the cells expressing the empty vector (E). This data represents three independent experiments.