Determination of the consequences of VHL mutations on VHL transcripts in renal cell carcinoma

Abstract

Genetic and epigenetic changes in the von Hippel-Lindau (VHL) tumour suppressor gene are common in sporadic conventional (clear cell) renal cell carcinoma (ccRCC). The effects on VHL expression are unknown but increased understanding may be relevant clinically, either in terms of prognosis or in therapy selection. We have examined the expression of VHL mutant RNA in 84 ccRCC tumours previously screened for mutations in genomic DNA, 56 of which contained 52 unique mutations or polymorphisms. Based on the predicted change to the primary amino acid sequence, 24 of the mutations were missense, 11 resulted in frameshifts with premature truncation, 9 resulted in immediate truncation at the site of the mutation and 2 were frameshifts which extended the reading frame beyond the normal stop codon. Nine tumours had intronic variants, including substitution of invariant residues at splice sites, deletion of nucleotides spanning the exon-intron junction, an intronic variant of unknown function and the polymorphism c.463+43A>G. Four variants were identified which were present in genomic DNA but not in mRNA. Three of these, all encoding apparent missense changes to the primary amino acid sequence, were located close to the ends of exons, reduced the strength of the splice site and function as null rather than missense variants. One nonsense variant was not detectable in mRNA but all other mutations resulting in premature truncation codons (PTCs) were, suggesting truncating VHL mutations may potentially generate truncated VHL protein. An intronic variant, c.341‑11T>A, previously regarded as of unknown function, is associated with an increased level of skipping of exon 2 and may, therefore, reduce production of pVHL. Our data show that the biological consequences of VHL mutations are not necessarily predictable from the sequence change of the mutation and that for the majority of VHL mutations, the potential for the generation of mutant protein exists.

Figure 1

Methylation of VHL promoter in cell line DNA. Eleven cell lines were examined for the presence of methylation at the VHL promoter using methylation-specific PCR. The methylated DNA reaction produces a PCR product of 158 bp from bisulphite-converted methylated DNA. The unmethylated DNA reaction produces a PCR product of 165 bp from bisulphite-converted unmethylated DNA. Neither reaction produces a product from DNA which has not undergone bisulphite conversion (data not shown). The CpGenome methylated DNA control contains both methylated and unmethylated DNA and produces a product with each reaction. All cell lines tested produced a product with only one of the two reactions and hence contain either only methylated or only unmethylated DNA.

Figure 2

Agarose gel images of RT-PCR products. (A) Schematic showing the two transcripts produced from the VHL gene. Amplification using primer set 3, in which the primers are located in exons 1 and 3, produces two products. The larger product, 1–3, (442 bp) is produced from full length VHL mRNA. The smaller product, Δ2, (319 bp) lacks exon 2. (B) Agarose gel images of RT-PCR products showing 1–3 and Δ2 products. In normal kidney tissue, the Δ2 product is much weaker than the full length product. The same pattern was observed in the majority of the tumour samples (data not shown). We observed four tumours in which the relative intensity of the bands was notably different. Tumour R69T had a clearly brighter lower band; in tumour R281T both bands were of similar intensity and in tumours R17T and R248T, although the full length band was stronger, there was less difference in intensity between the two bands than in control samples. Also shown are two tumours which contain the common polymorphism c.463+43 A>G. As this image is compiled from several individual gels, the absolute distance separating the two bands may differ but in every case the mobility relative to molecular weight markers and to controls was the same.

Figure 3

Mutations absent from VHL RT-PCR products. The left column shows sequencing traces of VHL RT-PCR products from seven tumours. The centre column shows sequencing traces of VHL PCR products amplified from the co-purifying DNA and shows the presence of the expected variant in each case compared to a wild-type control sample (right column). Exon-exon and intron-exon boundaries are indicated. Forward or reverse orientation of the trace is indicated and may differ between RNA and DNA sources. In panels A–D, each mutation is located in an exon and its absence from RNA was unexpected. In each case, the mutation is clearly present in DNA. In panel E, the mutation deletes the exon 1 donor splice site and mature mutant message was not expected. Again, the mutation is clearly present in tumour DNA. Panels F and G show data from two tumours in which a substantial difference in mutation level was observed between RNA and DNA. In each case, the mutation is present as the minor fraction in RNA but the major fraction in DNA.