Two single nucleotide polymorphisms in the von Hippel-Lindau tumor suppressor gene in Taiwanese with renal cell carcinoma

Von Hippel-Lindau (VHL) disease (OMIM 193300) is an autosomal dominantly inherited tumor syndrome affecting 1 in 36,000 in the human population [1]. The most common types of tumors are retinal angioma, cerebellar and spinal hemangioblastoma, clear cell renal cell carcinoma (RCC) and pheochromocytoma [2]. Tumors form when there is biallelic VHL tumor suppressor gene inactivation in a two hit model of tumorigenesis [3–5]. The VHL tumor suppressor gene is located on the short arm of chromosome 3 mapped to 3p25-3p26 and consists of 639 nucleotides in 3 exons encoding 213 amino acids [6, 7]. Germ-line mutations have been detected in approximately 500 VHL family members including deletions in part of or the whole gene, as well as intragenic point mutations and micro-deletions/insertions [8]. Somatic inactivating mutations have been identified in tumors from VHL-affected [9] and sporadic RCC [6, 10] patients.

The etiology of renal cancer is still not fully understood. Nevertheless, a number of environmental factors have been implicated in sporadic RCC [11]. RCC is the most common malignant renal cancer with around 7.5 cases per 100,000 people, which is equal to about 3% of all adult cancers in Western countries [12]. Most RCCs are classified as clear-cell RCC. More than half of RCC cases are linked to either VHL gene mutations or transcriptional repression with hypermethylation in the promoter and first exon regions [13, 14]. Multiple and bilateral RCCs are present in up to 40% of VHL patients. It is highly possible that subjects with VHL disease will develop RCC if they live long enough [15]. As early detection and intervention have reduced the death rate from VHL central nervous complications, bilateral multiple cystic RCC has become the leading cause of death in patients with VHL disease [16]. Recent reports have shown that acquired cystic disease-associated RCC frequently occurs with abnormalities on chromosome 3 in the Taiwanese population [17]. To date, no studies have focused on the association between VHL gene malfunction and RCC in Taiwan.

Two single nucleotide polymorphisms (SNPs), rs779805 and rs1642742 involving both A and G, located in the promoter and 3′ untranslated regions of the VHL gene are informative and implicated in the occurrence of RCC worldwide [18–20]. The aim of this study is to clarify whether these polymorphisms are associated with RCC in Taiwanese. We confirmed somatic mutation and loss of heterozygosity (LOH) at these two loci in RCC patients. In addition, a genetic association pilot study using these two SNPs for germline DNA genotyping was conducted to test the potential causality between VHL gene dysfunction and RCC susceptibility in the Taiwanese population.

All increases in rs1642742 shown in Table 2 and Table 3 are higher than in rs779805, indicating that rs1642742 is a more sensitive risk factor for sporadic RCC. However, further studies with a larger sample size are needed to clarify this phenomenon. Statistical significance was reached with just 19 samples for AG heterozygote frequencies in rs1642742 in the comparison of germline DNA between RCC patients and controls using chi-square test (χ² = 4.397, p = 0.036; bold in Table 2). Statistical significance was also reached in both loci between older and total RCC cases (χ² = 5.132, p = 0.023* for rs779805; χ² = 7.948, p = 0.0048* for rs1642742; bold in Table 3). AG heterozygote at rs1642742 was significantly associated with female gender and clear cell type (p = 0.018* and p = 0.028*; bold in Table 3). However, the small sample size of 19 patients in this study may not be enough to make broad generalizations.

Among the RCC patients in this study, the G allele carriage in rs779805 showed a slightly increasing trend. The location of rs779805 is 128 bp upstream from the major transcription-starting site within the promoter region of the VHL gene and its 5’ adjacent nucleotide is C. The somatic mutations from AA homozygote to AG heterozygote at rs779805 create a new CpG island that can be methylated to suppress gene expression. A similar rationale can be applied to RCC patients with germline AG heterozygote at rs779805. These somatic mutations were observed in four out of 15 RCC patients aged 50 or over (Table 1). The accumulation of somatic mutations becomes more evident late in life. Cancer may develop after shutting down of the function of VHL gene, when A allele is changed to G allele, followed by methylation of the CpG island.

Among the haploinsufficient tumor suppressor genes, it is possible that one allele is not able to function sufficiently [26]. Previous evidence regarding the Dmp1 gene has shown the effect of haploinsufficiency on tumorigenesis [27]. The differences in the distributions of AA homozygote and AG heterozygote at rs779805 and rs1642742 in this study may be a case of haploinsufficiency. GG homozygote was not observed in our RCC samples. A possible explanation is that GG homozygote causes early cell death before clinical cancer can develop.

Our results were consistent with those of previous reports, namely that VHL gene dysfunction is observed in sporadic RCC, as well as in hereditary cases [13, 28, 29]. Three polymorphic markers at 3p25-p26 have been examined for LOH in RCC [18]. Patients with sporadic RCC have been shown to have LOH in two SNPs in VHL gene [19, 20] and present with a biallelic polymorphism with either an A or a G at positions 19 (rs779805) and 1149 (rs1642742) in the nucleotide sequence [22, 23]. From a series of studies performed in Western countries, the G allele carriage at rs779805 and rs1642742 is an important risk factor for clear cell RCC [25], consistent with our findings. In this study, it was found that LOH at rs779805 and rs1642742 in the VHL gene in RCC patients manifests only late in life (Table 1). Our results were also consistent with previous findings of LOH in most RCC patients with AG heterozygosity [19, 20]. The current findings agree with Knudson’s two-hit theory that, in addition to gene mutation and hypermethylation, LOH is one of the major mechanisms resulting in tumor suppressor gene (such as VHL gene) inactivation [3–5]. LOH in tumor suppressor gene has been observed in most populations. However, ethnic differences may affect the frequency of occurrence [30]. LOH was considered positive when R was larger than 1.25 or smaller than 0.8 because even partial inactivation of tumor suppressors can contribute to tumorigenesis [5].

Linehan et al. reported that VHL gene inactivation might occur in the early stage of tumorigenesis in RCC [31]. In this study, most RCC patients with AG heterozygosity were identified as having LOH. LOH in VHL gene, therefore, is a beneficial tool for early diagnosis of RCC, as well as for monitoring of recurrence in RCC patients [31]. VHL gene polymorphism/association information obtained from this study is useful for further genetic RCC studies and for facilitating the development of new treatments. The results of this study can also be used to establish genetic diversity panels for the VHL tumor suppressor gene in the local population. Although further studies with larger sample sizes are needed to address the details of the association of VHL SNPs with RCC, as well as ethnic variation, these SNPs may be useful genetic tumor markers for the molecular diagnostics of clear cell RCC in the elderly population in Taiwan.

In the current study, we examined the association of two allelic VHL gene polymorphisms, rs779805 and rs1642742, with RCC in patients from Taiwan. By comparing the DNA from normal and tumor tissues, AA to AG somatic mutations at rs779805 and loss of heterozygosity at both rs779805 and rs1642742 in 10 out of 15 RCC patients aged 50 or over were observed. In comparison with the healthy control group, the G allele and AG heterozygote frequencies at these two loci were much higher in the patient germline DNA than in the control group on genetic association pilot study, especially for rs1642742 and late onset RCC, suggesting that the existence of G allele at both rs779805 and rs1642742 is of importance in RCC tumorigenesis. However, more comprehensive and detailed research is needed to address the clinical relevance. Larger sample size is required to determine the exact power of correlation between these two genetic polymorphisms and RCC.