Von Hippel-Lindau disease (VHL) (OMIM nr. 193300) is a rare autosomal dominant multi-organ disease caused by molecular abnormalities of the VHL tumor suppressor gene [1]. Patients with VHL are at risk for development of retinal, cerebellar, spinal, pancreatic and renal hemangioblastomas, pulmonary and liver hemangiomas, clear-cell renal carcinomas, pheochromocytomas, endolymphatic sac tumors, multiple renal, epididymal and pancreatic cysts, cystadenomas of the epididymis and of the broad ligament, and pancreatic islet cell tumors [1–3]. Based on the presence or absence of pheochromocytoma, two main subtypes of the VHL disease have been identified. Patients with VHL type 1 are at risk to develop renal cell carcinoma and hemangioblastoma but predominantly without pheochromocytoma, while those with VHL type 2 may have all manifestations of the disease predominantly including pheochromocytoma. VHL type 2 has been subdivided into subtype 2A and 2B, with a low and high risk of renal cell carcinoma, respectively, whereas subtype 2C is a pheochromocytoma-only phenotype. The prevalence of the VHL disease varies between 1:39,000 in Germany and 1:53,000 in East Anglia [4, 5]. Manifestations of the disease show variable expression and several patients may have only one manifestation [4].

The human VHL gene maps to chromosome 3p25-26 [1]. As predicted by Knudson's two-hit model, tumor development requires inactivation of both copies; the first hit, the germline mutation or deletion is followed by somatic alterations usually detected as loss of heterozygosity. The VHL gene has two translational initiation sites separated by 53 codons. Of the two proteins encoded by the VHL gene, the larger one contains 213 amino acids (pVHL30), whereas the shorter protein consists of 160 amino acids (pVHL18). Both proteins are functionally active and they share the same mechanism of tumor suppressor activity [6–8]. The VHL proteins form a multimeric complex with Elongin B, Elongin C, Cul2 and Rbx1. This complex is involved in ubiquitin-dependent proteolysis of large cellular proteins via controlled degradation of α-subunits of the heterodimeric transcription factor hypoxia inducible factor (HIF) [9, 10]. In addition, complexes containing pVHL, Elongin B, Elongin C, Cul2 and Rbx1 target proteins are involved in cell-cycle regulation for degradation, suggesting that pVHL has a possible role in cell-cycle exit [11]. In 1999, Stebbins and co-workers presented the three-dimensional protein structure of the VHL-ElonginC-ElonginB complex. They identified two functionally active sites; the ElonginC binding site in the α-helical domain involving residues between amino acids 157 and 170, and the HIF binding site in the β-sheet domain from residue 91 to 113 [8].

The majority of VHL patients have deletions, insertions, or mutations downstream of the second translational initiation site located at codon 54 of the VHL gene. Interestingly, missense mutations encoding residues of the protein binding sites have been associated with VHL type 2, whereas VHL type 1 is caused either by missense or nonsense mutations affecting the hydrophobic core, or by partial gene deletions which result in a complete defect of protein function. It has been also demonstrated that some mutant pVHL, which are associated with the type 2C phenotype, may impair fibronectin matrix assembly while they retain the ability to down regulate HIF [12].

Because mutations affecting the first 53 codons of the VHL gene have no effect on the structure of the shorter VHL protein, it seemed particularly interesting to clarify whether these mutations could exert a pathogenic effect. In one study the Pro25Leu variant has been associated with a sporadic pheochromocytoma [13]. but other studies involving a limited number of cases failed to confirm the pathogenic role of this variant [14, 15]. In this paper we report a large Hungarian VHL type 2 family with a disease-causing AGT80AAT (Ser80Ile) c.239G>A, p.Ser80Ile mutation and a concurrent CCT25CTT (Pro25Leu) c.74C>T, p.Pro25Leu variant (identification number: rs35460768 (dbSNP127) of the VHL gene. We show that in family members the Ser80Ile mutation, but not the Pro25Leu variant co-segregated with the disease. In order to confirm the pathogenicity of the Ser80Ile mutation and to test whether the Pro25Leu variant might be neutral, an evolutionary multiple sequence alignment analysis (MSA) combined with a Align GVGD was.performed. Three-dimensional modeling of the Ile80-mutant protein is also presented.

In this paper we describe a large Hungarian VHL type 2 family consisting of 32 members, in whom a disease-causing AGT80AAT (Ser80Ile) c.239G>A, p.Ser80Ile mutation and a concurrent CCT25CTT (Pro25Leu) c.74C>T, p.Pro25Leu variant were identified. Screening of family members indicated that 7 family members had the Ser80Ile mutation and 2 members had the Pro25Leu variant, and that the two genetic alterations were transmitted in separate alleles. More importantly, we showed that the Ser80Ile mutation, but not the Pro25Leu variant co-segregated with the VHL type 2 phenotype and that the Pro25Leu variant never occurred in family members who had manifestations of the disease. However, one family member who had both genetic alterations and one member with the Ser80Ile mutation without the Pro25Leu variant showed no manifestations of the disease, suggesting that this variant is not pathogenic for VHL.

In order to predict the pathological role of the Pro25Leu and Ser80Ile variants, first we constructed a multiple sequence alignment using evolutionary distant species. It has been shown previously that the addition of more distantly related sequences is essential for a more accurate prediction (20). However, in our case this approach revealed that both variants might be neutral, which is in contrast with earlier clinical findings suggesting the disease causing role of the Ser80Ile but not of the Pro25Leu. The Ser80Ile has been previously reported only in one VHL patient without clear cell renal carcinoma [23], but several other missense mutations at codon 80 have been described. The Ser80Gly mutation has been detected in a 12-yr-old patient with bilateral pheochromocytomas and multiple congenital malformations [24] and in a patient with pheochromocytoma [25]. The Ser80Asn mutation was identified in a Slovakian family with VHL type 1 phenotype including pancreatic islet tumor in one affected individual [26]. In addition, the pathogenic role of Ser80Arg [27, 28] and Ser80Asn mutations has been also documented in VHL patients [25]. Our study shows for the first time that the Ser80Ile mutation is associated with bilateral pheochromocytoma presented as a first manifestation of the disease, which points out that VHL patients harboring this mutation require careful screening for pheochromocytoma.

The association between various missense mutations at codon 80 of the VHL gene and VHL disease in previous studies and ours is not unexpected, since our evolutionary alignment analysis showed that in this position the Ser is a highly conserved amino acid residue among different species. Moreover, computational protein modeling of a change of Ser to Ile at position 80 indicated potentially important consequences on three-dimensional structure of the pVHL. Ser80 is a polar amino acid located in the hydrophobic core of the β-domain close to the α-β interface of pVHL and ElonginC interaction. According to our protein modeling, the change of Ser to a larger and nonpolar Ile at amino acid position 80 results in a complex rearrangement of the three-dimensional structure of the protein binding interface, which disturbs HIF1α and fibronectin binding [29]. Miller and co-workers have already demonstrated that VHL gene mutations within L1 and L7 loops distrupt the dynamic coupling of the pVHL and HIF-1α and that this may be an important mechanism for the development of tumors [30]. Other studies using three-dimensional modeling and a computer algorithm FOLDEF provided a quantitative estimation of the importance of the interactions contributing to the stability of proteins and protein complexes [31]. In the case of the Ser80Ile, the free energy difference between the wild type and mutant proteins was 12.2 kcal/mol, which is higher than the previously established cut-off value for the development of clear-cell renal carcinoma or pheochromocytoma [32].

Although mutations affecting the first 53 codons of the VHL gene have no effect on the structure of the shorter VHL protein, a few mutations at codons 25, 38, 46 and 52 have been considered to exert a pathogenic role. The Pro25Leu and Ser38Pro have been identified in patients with pheochromocytomas, whereas the Glu46Stop and Glu52Lys have been associated with VHL disease [32, 27]. However, Rothberg et al. reported a VHL patient with two VHL gene mutations, Pro25Leu and Pro86Arg, in whom the Pro86Arg was considered more likely to be pathogenic than the Pro25Leu based on an allelic frequency of 0.5% of the latter variant in anonymized DNA samples [14]. This Pro25Leu variant was detected in other healthy VHL family members from Poland and North America [33, 14]. In agreement with these observations, the Pro25Leu carrier in our family had no manifestations of the disease, suggesting that this variant does not represent a disease-causing mutation.

The Ser80Ile mutation of the vhl gene in a large Hungarian kindred was found to be associated with VHL type 2 presenting with both pheochromocytoma and renal cell cancer. Therefore, patients with this mutation require careful surveillance, including search for pheochromocytoma. The Pro25Leu variant in this family apeared to be neutral, but its co-existence made genetic counseling difficult.