A family with VHL syndrome from a Han Chinese population was recruited for the present study in Hunan Cancer Hospital (Changsha, China) between May 2014 and June 2014. There were originally 23 patients in this pedigree; however, 6 patients succumbed to mortality prior to recruitment. In total, 17 subjects [including 8 males and 9 females; aged 4–65 years old; one patient suffering from VHL syndrome (proband)] were enrolled and underwent detailed clinical examinations, including the Romberg sign test, in which the standing patient is asked to his/her close eyes with their heels together and if the patient loses balance, a positive result is recorded (15). In addition, finger-nose tests were performed, which requires the patient to alternately touch his/her nose and the examiner's finger as quickly as possible. If the patient fails to touch his/her nose accurately, a positive result is recorded (15). All members of the family were examined by two experienced surgeons. Diagnoses of VHL syndrome were determined via investigation of medical histories and performance of physical examinations, including a complete neurological examination and brain magnetic resonance imaging (MRI). In addition, 200 unrelated volunteers (aged 35–65 years old; 116 males and 84 females) that did not have VHL syndrome or present any associated phenotypes were recruited as controls in the Health Management Center, Xiangya Hospital of Central South University (Changsha, China) between September 2014 and October 2014. The present study was approved by the Institutional Review Board of the Xiangya Hospital of Central South University, and all subjects enrolled in the present study provided written informed consent prior to examination.

Biopsy tissues isolated from the intracranial cyst of the proband were obtained via surgical excision, fixed in 4% paraformaldehyde at room temperature for one week and then embedded in paraffin. Histopathological analysis of paraffin-embedded biopsy tissue sections (6 µm) from the proband was performed using a Hematoxylin and Eosin Staining Kit (cat. no. M020; GEFAN Biotechnology, Co., Ltd., Shanghai, China) (16). Sections were blocked with 5% horse serum (cat. no. HQ90041; Shanghai Bangyi Biotechnology Co., Ltd., Shanghai, China) at room temperature for 30 min and subsequently incubated at 4°C overnight with antibodies against glial fibrillary acidic protein (GFAP; 1:200; cat. no. KIT-0031), epithelial membrane antigen (EMA; 1:80; cat. no. KIT-0011), S-100 protein, E3 SUMO-protein ligase nse (NSE; 1:100; cat. no. MAB-0584), hematopoietic progenitor cell antigen CD34 (CD34; 1:50; cat. no. KIT-0004), Factor VIII-related Antigen (F8; 1:100; cat. no. RAB-0070) and proliferation marker protein Ki-67 (Ki-67; 1:100; cat. no. MX006) (16). All primary antibodies were purchased from Fuzhou Maixin Biotech Co., Ltd. (Fuzhou, China). Following this, sections were incubated with horseradish peroxidase-conjugated anti-mouse/rabbit secondary antibodies (working dilution was provided by the kit; cat. no. KIT-5010; Fuzhou Maixin Biotech Co., Ltd.) at room temperature for 2 h. Images of the sections were captured using Photographs were taken using a light microscope Olympus BX51 (Olympus Corporation, Tokyo, Japan; magnification ×400) and a Nikon N6006 camera (Nikon Corporation, Tokyo, Japan). The number of positive cells was calculated as the total area of positive staining divided by the area of an individual positive cell using Image Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). The percentage of positive cells was calculated as the number of positive cells divided by the total number of cells.

Genomic DNA was isolated from peripheral blood lymphocytes of all subjects using the standard phenol-chloroform method (17). VHL was amplified via polymerase chain reaction (PCR). Three pairs of primers were designed to cover all of the exons and splicing junctions of VHL (Table I). The thermocycling conditions of PCR used were as follows: Initial Denaturation at 95°C for 30 sec; followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 58°C for 30 sec and elongation 72°C for 30 sec; and a final extension at 75°C for 5 min. PCR amplification was performed using an ABI 2720 PCR system (Thermo Fisher Scientific, Inc., Waltham, MA, USA) using a 10 µl mixture containing premixed DNA polymerase (5 µl; Takara Biotechnology Co., Ltd., Dalian, China), primers (2 µl), genomic DNA (1 µl) and double distilled H₂O (2 µl). PCR products were detected using 6% polyacrylamide gel electrophoresis with silver staining. Sanger sequencing was performed using an ABI 3100/3130 Genetic Analyzer automated sequencer (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The sequencing results were analyzed using SeqMan 3.0 (DNASTAR, Inc., Madison, WI, USA), and compared with the reference sequences listed in the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/). Mutation naming followed the nomenclature recommended by the Human Genomic Variation Society (http://www.hgvs.org/).

Total RNA was isolated from lymphocytes obtained from the patient using a GeneJET RNA Purification Kit (Fermentas; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. First strand complementary (c)DNA was synthesized from total RNA at 42°C for 60 min and then at 70°C for 5 min using a RevertAid First Strand cDNA Synthesis Kit (Fermentas; Thermo Fisher Scientific, Inc.), according to the manufcaturer's instructions. Primer sequences for semi-quantitative (sq)PCR were designed using Primer 3 (version 4.1.0; http://primer3.ut.ee/) and were as follows: pVHL forward, 5′-CTCCCAGGTCATCTTCTGCA-3′ and reverse, 5′-CTTGACTAGGCTCCGGACAA-3′. sqPCR procedures were performed according to the following protocol: Initial denaturation at 95°C for 30 sec; followed by 10 cycles at 95°C for 30 sec, 62°C for 30 sec and 72°C for 30 sec; a further 23 cycles at 95°C for 30 sec, 59°C for 30 sec and 72°C for 30 sec; and a final extension at 75°C for 5 min. Following this, 5% agarose gel electrophoresis was performed to verify the PCR products. Proteins were visualized using ethidium bromide. Normality of distributions was determined using the Shapiro-Wilk test. Statistical significance was assessed with an unpaired two-tailed Student's t-test, using GraphPad Prism 6.0 (Graphpad Software, Inc., La Jolla, CA, USA). Data are presented as mean ± standard deviation.

Lasergene MegAlign 9.0 (DNAStar, Inc.) was used to investigate whether the identified mutation of pVHL was conserved between different species. MutationTaster (http://mutationtaster.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/index.shtml) and SIFT (http://sift.jcvi.org) software, all available online, were selected to investigate the effect of the mutated amino acid on the protein's structure and function. SWISS-MODEL (https://swissmodel.expasy.org) and PyMol software (version 1.5.0.3; DeLano Scientific, San Carlos, CA, USA) were used to predict and visualize three-dimensional protein models.

A four-generation family with diagnosed VHL syndrome was recruited for the present study. The family consists of 23 individuals, including 11 males and 12 females (Fig. 1). The proband was a 28-year-old male, who presented with an episodic headache for 10 years. Both the finger-nose test and the Romberg's sign were recorded as positive in the physical examination. A cyst and a nodule were revealed via brain MRI (Fig. 2). The cyst was demonstrated to be a hemangioblastoma tumor following surgical excision followed by histopathological analysis of the biopsy tissues. H&E staining revealed that the lesion contained a large number of thin-walled capillaries (Fig. 3). The sole neoplastic element, known as the ‘stromal cell’, inhabited the interstices between ramifying vascular arcades. Stromal cells stained for S-100 and NSE exhibited dense-core intracytoplasmic granules, however did not demonstrate expression of GFAP or EMA (Fig. 3), which has previously been detected immunohistochemically in most nonneoplastic epithelia (18). Stromal cells did not exhibit positive staining for endothelial markers such as CD34, whereas the thin-walled capillaries were positive; stromal cells occasionally exhibited the expression of vascular marker F8 (Fig. 3). The expression of Ki-67, which is a proliferation marker (19), was <1% (Range of normal value 1–2%; Fig. 3). Therefore, the proband was diagnosed with VHL syndrome at the Hunan Cancer Hospital (Changsha, China). A further five affected members of the family had previously died from this syndrome.

A heterozygous mutation c.345C>A (p.H115Q) in exon 2 of the VHL gene was revealed to be present in the proband III:2 (Fig. 4A). In addition, two healthy individuals (III:4 and III:8) in the pedigree were demonstrated to carry the mutation, as revealed by Sanger sequencing (Fig. 1). The mutation was not present in the 200 healthy patients.

Alignment of VHL amino acid sequences in numerous species revealed that the histidine at the 115th amino acid site is highly conserved (Fig. 4B). The functional prediction software packages Polyphen-2, SIFT and MutationTaster; demonstrated that the variant is likely to be deleterious (data not shown). The structures of pVHL and p.H115Q, as predicted using SWISS-MODEL, were markedly different from one another: The wild type pVHL exhibited an imidazole ring structure (Fig. 4Ca), and the p.H115Q mutant exhibited a non-imidazole ring structure (Fig. 4Cb). Protein surface analysis revealed marked changes between the wild type pVHL and the p.H115Q mutant pVHL. In wild type pVHL, the area highlighted by the red arrow is non-polar charged and forms a continuous hydrophobic cleft, which facilitates its interaction with HIF-α (Fig. 4D). However, the H115Q mutant introduces a negative charge in this area (right panel), subsequently disrupting the hydrophobic environment required for binding with HIF-α (Fig. 4D).

cDNA was generated by reverse transcribing RNA extracted from peripheral blood samples obtained from patients in the pedigree and the healthy controls. No significant difference in the expression of pVHL was demonstrated between the mutated and wild type cDNA, as demonstrated by RT-sqPCR analysis (Fig. 5).