Von Hippel-Lindau (VHL) disease (OMIM 193300) is an autosomal dominant familial cancer syndrome that increases susceptibility to several benign and malignant tumors, including central nervous system (CNS) hemangioblastomas, renal cysts and renal cell carcinoma (RCC), as well as pheochromocytoma (PCC). Additional tumors, such as pancreatic and endolymphatic sac tumors and epididymal and broad ligament cystadenomas, have also been previously described (1–6). VHL syndrome accounts for ~1/3 of patients with CNS hemangioblastoma, >50% of those with retinal angioma, 1% of those with RCC, 50% of those with apparently isolated familial PCC and 11% of those with an apparently sporadic PCC (7–9). VHL is present at a frequency of 1 in 36,000 live births in Eastern England (2,5). Similar to other hereditary syndromes, VHL has its own clinical classification based on genotype-phenotype associations. Based on clinical symptoms, patients with low-risk PCC (<5%) are classified as VHL type 1 and those with high-risk PCC (>60%) as VHL type 2. VHL type 2, according to 7–20% of VHL kindreds, is further subdivided into three hypotypes, 2A (patients with a low risk for RCC), 2B [patients with an increased risk (~70%) for RCC] and 2C (affected members with isolated PCC) (1,2,10).

VHL, the gene responsible for VHL disease, was identified in 1993 (11). It is a pleiotropic tumor suppressor gene located on chromosome 3 (3p25.3) that is widely expressed in fetal and adult tissues (11,12). Several VHL germline mutations have been identified in individuals with VHL disease, as well as somatic mutations in sporadic tumors (5,13–16). VHL type 1 families frequently harbor larger deletions and nonsense mutations in addition to missense variants in the VHL gene, and patients with type 2 disease carry missense mutations almost exclusively (5,10). Up to 96% of patients with PCC have missense mutations (10).

The first VHL case with a germline mutation in China was reported by Huang et al(17) in 2004. In the present study, two frequent VHL germline mutations, c.A233G (p.N78S) and c.G482A (p.R161Q), are described in two families with type 2A and 2C VHL syndrome. Patients underwent surgery and were subsequently followed up.

In family 1 with type 2A VHL syndrome, we identified a heterozygous variant, c.A233G (p.N78S), within exon 1 of the VHL gene in two affected cases and one clinically normal family member (F1-III-1, the proband's 5-year-old son; Fig. 3A). A heterozygous nucleotide substitution within exon 3 of the VHL gene, c.G482A (p.R161Q), was identified in the proband (F2-II-11) and two additional cases (F2-III-4 and F2-III-9) of family 2 with VHL type 2C (Fig. 3B). No mutants of the RET gene were identified in these cases.

VHL disease-related PCC is a tumor of the main sympathetic paraganglion, the adrenal medulla, which is complicated by hypertension. PCC is usually benign, with ~20% malignant cases (18). Molecular genetic studies suggest that ~50% of individuals with apparent familial non-syndromic PCC have VHL germline mutations (18). Previous studies have shown that PCC occurs sporadically or as part of hereditary tumor-predisposition syndromes caused by germline mutations affecting one of the ten following susceptibility genes: VHL, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, NF1, TMEM127 or MAX (Table II) (1,2,19–27). The majority of familial cases of PCC and 10–20% of sporadic cases carry germline mutations of these genes (4). RCC represents 90–95% of neoplasms arising from the kidney, which is an important cause of death in VHL disease (7). RCC is rare in children and young adults, and occurs in both a nonfamilial and familial form. Hereditary RCC accounts for ~4% of RCC, including the following disorders: VHL disease; hereditary leiomyomatosis related to FH gene mutation; tuberous sclerosis, which results from the germline mutation of TSC1 and TSC2 genes; hereditary papillary renal cell carcinoma (HPRCC), which is associated with MET gene; and the gene responsible for Birt Hogg-Dubé syndrome, which has been reported to be the FLCN gene (Table II) (28–31). In a familial form, RCC occurs at an early age (with the exception of HPRCC) and usually presents bilaterally with a high incidence (7,31). According to a recent study, 20/255 patients (7.8%) with hereditary leiomyomatosis and RCC were reported to have primary adrenal lesions, including 4 patients with bilateral adrenal lesions and 4 with multiple nodules (28). Therefore, in future studies on PCC and RCC patients, targeted regions (all exons of VHL, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, NF1, TMEM127, MAX, FH, TSC1, TSC2, MET and FLCN) or exome sequencing should be analyzed (32).

The VHL gene has been conserved throughout evolution and is found in all multicellular organisms. The two VHL gene products, pVHL, a 30-kDa full-length protein [p30, 213 amino acids, NM_000551.2 (variant 1 mRNA)], and a 19-kDa protein [p19, 160 amino acids, NM_198156.1 (variant 2 mRNA)], suppress tumor formation in nude mice and regulate hypoxia-inducible factor-α (HIF-α) (33,34). pVHL has two domains, the β domain (N-terminal β-sheet domain), which contains a macromolecular binding site, and an α domain (N-terminal α-helical domain), which interacts with elongin C (35). The region between codon 75–82 is located in the β-binding domain of VHL protein; thus, the p.N78S mutation interferes with HIF binding, which results in the excessive transcription of HIF target genes, such as vascular endothelial growth factor (13). Codon 161 is located in the α domain and interacts with elongin C. The mutation in the α domain affects VHL tumor suppressor activity (36,37). Through errors in chromosomal replication during cell division, additional genes or modifying factors may be involved in the genetic basis of VHL (37).

VHL genotype-phenotype correlations have been previously established (5,17). The p.N78S mutation has been reported to be associated with PCC, RCC and CNS hemangioblastomas (8). The p.R161Q mutation was first reported by Tong et al(38) in Chinese VHL patients who had non-syndromic familial PCC without hypertension and is also known to be associated with retinal and CNS hemangioblastoma and RCC (10). For patients included in the present study, VHL mutations were consistent with the type 2A and 2C phenotypes. In particular, p.R161Q was associated with PCC with hypertension syndrome, and p.N78S had a more severe clinical phenotype (RCC and bilateral PCC) compared with p.R161Q. However, none of the mutations were associated with CNS hemangioblastomas.

The identification of the VHL tumor suppressor gene facilitates the diagnosis of VHL disease, particularly in patients who do not meet the clinical diagnostic criteria. It also has important implications on the estimation of risk for the development of further tumors in patients and of VHL disease in their relatives. In family 1, a young family member (F1-III-1, 5 years old) did not have an evident clinical phenotype but carried the p.N78S mutation. The identification of the VHL tumor suppressor gene is beneficial for the early prevention and treatment of VHL syndrome. Additionally, the mean age at diagnosis of PCC and RCC in VHL is ~30 and ~40 years, respectively (39,40). However, according to a clinical report, a 2.75-year-old female patient with PCC was found to carry a p.R161Q germline mutation (40). This is the youngest patient with PCC reported to date. For patients in the present study, the age of onset for patient F2-III-9 was 9 years, while the other two patients were 18 and 28 years old when they were diagnosed with PCC. Thus, molecular genetic testing and genetic counseling for patients with PCC and RCC should be performed at an early age. Early diagnosis of VHL would be beneficial, since regular surveillance for tumors and early intervention are suggested to result in better prognosis.

Numerous studies have shown that VHL patients treated with NSS have a high incidence of local recurrence of new primary tumors, but a low risk of distant metastasis. Approximately 25% of VHL patients with more advanced RCC (>3 cm) develop metastatic disease (41,42). Radical bilateral nephrectomy should be performed when RCC >3 cm, and renal transplantation is required. Bilateral PCC is usually treated by CSA (adrenal tumorectomy or partial adrenalectomy). Patients may avoid hormone replacement, but there would be the risk of late recurrence. Total adrenalectomy may be considered as an alternative when there is a high incidence of multiple bilateral adrenal masses and large tumors; however, life-long steroid replacement is required. All the patients should be followed up regularly to aid the identification of the processing adrenocortical insufficiency or Addisonian crisis (3).

In the present study, the p.N78S mutation patient (F1-II-1) with multiple bilateral RCC (1.0 and 1.0 cm) and right PCC (4.1 cm) was subjected to right NSS and right CSA. The left RCCs were observed using imaging and exhibited no marked enlargement, while locoregional recurrence, distant metastasis and/or CNS were not found in two cases (F1-II-1 and F1-II-3) 39 and 6 months after the surgery, respectively. Among the three p.R161Q mutation patients, one (F2-III-9) had a left PCC 8 years after presenting with a right PCC at 9 years of age, one (F2-III-4) had simultaneous bilateral PCC and the other (F2-II-11) only presented with a right PCC at 18 years of age. All of them underwent bilateral or unilateral CSA and adrenocortical function was normal without steroid replacement. Therefore, identification of the underlying mutation not only confirms the diagnosis, but also provides useful information for genetic counseling and subsequent management decision making.