Three novel germ-line VHL mutations in Hungarian von Hippel-Lindau patients, including a nonsense mutation in a fifteen-year-old boy with renal cell carcinoma

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited highly penetrant tumor syndrome affecting 1 in 36,000 individuals worldwide. VHL disease predisposes to retinal and central nervous system hemangioblastomas (HB), renal cell carcinoma (RCC), phaeochromocytoma, pancreatic endocrine tumors, endolymphatic sac tumors among other less frequent complications. Type 1 diseases is accompanied with low risk of phaeochromocytoma, type IIa is associated with high risk of phaeochromocytoma and low risk of renal cell carcinoma, while type IIb is linked to high risk of both phaeochromocytoma and renal cell carcinoma. In type IIc VHL disease only phaeochromocytoma develops. Patients carry a heterozygous germline mutation in the VHL gene. Tumor development is initiated by the somatic inactivation or loss of the remaining wild type VHL allele [1, 2]. VHL protein associates with the elongins B and C, cullin2 and Rbx and functions as the substrate recognition component of an E3-ubiquitin ligase that ubiquitinates HIF-1aplha under normoxic conditions resulting in HIF-1alpha proteolysis [3].

The protein model of pVHL19 contains two functional subdomains; the beta domain (residues 63–154 and residues 193–204) and the helical alpha-domain (residues 155–192). Two important binding sites within VHL protein have been identified; one responsible for elongin C binding in the alpha domain (amino acid residues 157–170) and the other in the beta domain responsible for the binding of HIF1alpha (amino acid residues 91–113) [4]. The majority of disease-causing missense VHL gene mutations are located in one of these two binding sites. Molecular genetic defects identified in the background of the disease have greatly helped to understand the role of VHL protein in the hypoxia sensing pathway and to recognize genotype-phenotype correlation, a prerequisite of efficient genetic counseling and patient follow-up [5]. More than 800 different germline mutations are listed in the VHL mutation database (http://​www.​umd.​be:​2020/​) [6] and also in a comprehensive analysis based on 945 VHL kindreds [7]. Here, we describe 7 members of 5 families with three novel and one previously reported point mutations in the VHL gene and correlate genetic findings with clinical phenotype.

Molecular genetic analysis of 11 members of 5 unrelated VHL families detected 3 novel and 2 previously described point mutations in the VHL gene. No alterations could be detected in the VHL gene with the MLPA test. All mutations associated to type I VHL disease. None of the mutations could be observed in one hundred control subjects. Phenotype characteristics and genetic data of the study population are summarized in Table 1. Electropherograms of corresponding VHL mutations are demonstrated in Figure 1.

Two affected probands of family B, sister (index patient, IP) and brother, with a positive family history of VHL disease were examined. The sister developed a retinal angioma of the right eye at the age of 48 years. MRI examination revealed a cerebellar hemangioma in both patients. RCC could not be revealed so far in any of the patients. Molecular genetic analysis detected the novel c.232A > T (p.Asn78Tyr) missense mutation in heterozygous form in both patients.

Family C consisted of two parents and their two daughters. The 12-year-old daughter (IP) was investigated because of a retinal angioma. A spinal cord hemangioma was detected on the craniospinal MRI. No other manifestations of VHL disease have been detected so far in her case. Radiological investigations detected bilateral kidney tumors, a cerebellar and a spinal cord hemangioma in her 34-year-old father. Histological analysis of the kidney verified RCC. Four years later, retinal angioma developed in both of his eyes. The mother and the other daughter were free from any manifestations of the disease. Both affected family members were heterozygous for the c.340 + 1 G > A mutation, located at the first exon-intron junction. The mutation destroys the conserved intronic donor splice site and most likely leads to premature protein termination (p.Gly114AspfsX6). The mother and the healthy daughter showed no mutations in the VHL gene. As in our case, the mutation associated to type I VHL phenotype in a previous report [19].

The 25-year-old male patient (Patient D) was the only available member of the family. His father had died several years ago due to a central nervous system tumor, probably a complication of VHL disease. The patient was diagnosed with cerebellar and spinal cord hemangioma and a subsequently occurring RCC, while other manifestations of VHL disease could not be detected. The novel heterozygous nonsense amber mutation (c.555C > A) was detected in Patient D. The mutation caused a previously reported protein truncation (p.185TyrX) [6, 20, 21].

Patient E suffered from retinal angioma and cerebellar hemangioblastoma. No other VHL-related manifestations could be revealed so far. Genetic analysis detected the previously described c.583C > T mutation resulting in premature protein termination (p.195GlnX). Interestingly, the mutation has been reported to associate to type I and type II VHL disease as well [20, 22, 23].

We have investigated seven VHL patients from five unrelated Hungarian families. After detailed physical, ophthalmological, radiological and laboratory investigations all patients were found to have type 1 VHL disease. Molecular genetic investigations detected three novel and one previously described point mutations in the VHL gene. According to the present and a previous report [24], there are 12 VHL families in Hungary identified so far. Nonsense, missense, frame shift mutations and exon deletions were detected in four, four, two and two families, respectively. These proportions show no major deviation from those of other populations and are comparable to proportions reported by the comprehensive analysis of Nordstrom-O’Brien et al [7]. The novel c.232A > T (p.Asn78Tyr) missense mutation associated to type I VHL phenotype. The site is evolutionary conserved suggesting it is an important amino acid position to maintain protein structure and function. Other mutations at the same codon, namely p.Asn78His, p.Asn78Ser, p.Asn78Thr and p.Asn78Ile associated with type I disease phenotype as well [20, 22, 23, 25‐29]. This mutation site is buried within the protein core where mutations were found to associate with lower risk of phaeochromocytoma compared to protein surface missense mutations [23]. N78H, N78I and N78S missense mutations were all reported to coincide with RCC, however the N78T mutation did not show this association [20, 22, 23, 25‐29]. According to a later publication RCC associates with high risk to missense mutations located between codon 74 and 90 [30]. The effect of the novel p.Asn78Tyr mutation on VHL protein structure was assessed using molecular modeling. The Asn78 side chain participates in the stabilization of the turn where it can be found. The breaking of the loop-stabilizing H-bond interaction between Asn78 and Arg82 as well as the replacement of this residue with a larger one obviously destabilizes this turn (Figure 3). It can cause partial or global misfolding. The most remarkable effect of this structural reorganization on protein interactions is the high probability of dissociation of the VHL and HIF-1alpha proteins. Despite the turn deformation, disruption of the VHL-Elongin C interaction could not be observed during the molecular dynamics simulation (Figure 3). In vitro analysis of pVHL mutants showed reduced ability to bind and ubiquitinate HIF-1alpha in case of type I, IIa and IIb mutations, on contrary, mutations associated to IIc phenotype showed normal binding and ubiquitination of HIF-1alpha protein. Completely absent HIF-1alpha binding was only observed in consequence of mutations associating to RCC in this study [31]. However, a recent computational study could not confirm the dose-dependent effect of VHL-HIF-1alpha dissociation and suggests that the canonical configuration of the wild-type beta domain is vital for the efficient functioning of the complex and that mutation of any of the residues implicated in the H-bond network in the binding site disrupts HIF binding [32]. Taken together, mutations in the beta domain disrupting the VHL-HIF-1alpha interaction lead to type I disease phenotype with an increased risk of RCC. Based on these data and our molecular modeling results we can conclude that the novel p.Asn78Tyr mutation associates to type I disease and has a high chance to associate to RCC as well, while only minor risk to associate to phaeochromocytoma. In an attempt to assess the effect of all previously reported missense mutations in familial VHL, we applied SIFT analysis and found that in type II disease the rate of tolerable mutations is significantly higher than in type I mutations. This finding further supports that deleterious mutations disrupting protein integrity are more prone to cause type I phenotype. Besides, it suggests that SIFT analysis can be a useful tool when quick prediction of a novel mutation is necessary.

As expected, nonsense mutations associated with type I VHL phenotype. Two of the three nonsense mutations associated to RCC as well. Previous publications show that large germline deletions, nonsense and frameshift mutations associate with type I VHL phenotype [6, 20‐22, 25, 33]. Consistent with previous publications indicating that RCC coincides more frequently with nonsense mutations than missense mutations, RCC only associated to MLTP in our patients [23, 34]. Nonsense mutations independent of their exonic location associate with earlier age at onset and higher age-related risk of RA and RCC compared to missense mutations and large deletions [23]. Supporting this observation, the patient with the p.55GluX mutation was 15-years-old when bilateral retinal angioma and bilateral RCC was diagnosed, which represents the earliest occurrence of RCC in VHL disease reported so far [1, 35]. VHL screening guidelines [2, 8‐10] recommend a screening schedule for VHL patients regardless of their genetic status in order to recognize VHL complications in a curable stage. The earliest occurrence of RCC was reported in a sixteen-year-old boy. Accordingly, screening guidelines recommend yearly or biannual abdominal MRI from sixteen years of age and yearly abdominal ultrasound starting at 8 years of age. The occurrence of RCC in a fifteen-years old boy with a truncating germline VHL mutation highlights that renal cell carcinoma can be detected in patients with nonsense VHL mutations younger than sixteen years of age. Taken into account that abdominal MRI scans with a higher resolution can detect RCC earlier than ultrasound, patients with nonsense mutations known to associate to RCC and early onset of the VHL disease [23] could be considered for earlier MRI scans. In general, genetic status might be considered to help tailoring individual screening schedules in the future.

Genotype-phenotype correlation was analyzed and related to the findings reported in previous publications. In our study population we confirmed that the disruption of the VHL protein integrity leading to the disregulation of HIF-1alpha associate to type I phenotype. Accordingly, we also showed that the proportion of non-deleterious mutations is higher in type II than in type I missense mutations among mutations reported in familial VHL cases. Here we show the earliest occurrence of RCC reported in VHL disease so far and suggest individual tailoring of screening schedule based on molecular genetic status. Clinical and genetic data presented in our study can help the routine of genetic counseling and patient follow-up, as well as the presymptomatic molecular genetic diagnostics and contribute to a better understanding of genotype-phenotype correlations.