Clinical and functional properties of novel VHL mutation (X214L) consistent with Type 2A phenotype and low risk of renal cell carcinoma

Sorrell AD, Lee S, Stolle C, Ellenhorn J, Grix A, Kaelin Jr WG, Weitzel JN. Clinical and functional properties of novel VHL mutation (X214L) consistent with Type 2A phenotype and low risk of renal cell carcinoma. This report describes clinical characteristics in families with a Type 2A phenotype and functional properties of a novel von Hippel Lindau variant (X214L). Pedigrees were analyzed. Analysis of von Hippel Lindau (VHL) coding exons and flanking intronic sequences in DNA from a proband with pheochromocytoma and islet cell tumor was performed. Western blot assays for VHL protein (pVHL), HIFα, and Jun B were conducted using VHL null renal clear carcinoma cell lines that were engineered to produce wild-type or X214L mutant pVHL. Pedigree analysis indicated that the variant tracked with disease and the same or similar VHL point mutations were identified in several Type 2A families. The predicted 14 amino acid extended pVHL variant, when reintroduced into VHL null cells, was stable and retained the ability to downregulate HIFα in a hydroxylation-dependent manner. In contrast, the variant was defective with respect to downregulation of JunB. pVHL X214L, like other pVHL variants associated with a low risk of clear cell renal carcinoma, largely preserves the ability to downregulate HIF. In contrast, this variant, like other pVHL variants linked to Type 2A disease, fails to suppress JunB. This underscores that JunB may play a role in the pathogenesis of Type 2A VHL disease.


Introduction
Von Hippel Lindau (VHL) syndrome is a rare autosomal dominant disorder that causes an array of potentially disabling and life-threatening tumors in children and adults. Deleterious mutations in the VHL gene cause tumors such as hemangioblastomas (HB) of the retina, cerebellum, brainstem, and spine; clear renal cell carcinomas (RCCs); pheochromocytomas (Ph); neuroendocrine tumors (N); endolymphatic sac tumors (ELST); cystadenomas of the epididymus, broad ligament, and pancreas; and cysts in the kidneys and pancreas (1,2).
RCC affects approximately 40% of VHL patients, and metastatic RCC is the most common cause of death in VHL germline carriers (3)(4)(5). VHL disease penetrance is age dependent with clinical symptoms developing in 19% of affected individuals by 15 years of age, 52% by age 25, and 91% by the age of 45 (6). The age-related penetrance and the specific types of tumors that develop in affected individuals depend upon the functional consequences of the specific VHL germline mutation.
The VHL tumor suppressor gene is comprised of three exons (639 nt), located on the short arm of chromosome 3 (3p26-p25; OMIM 608537). The VHL gene encodes for a protein comprised of 213 amino acids (24.2 kDa) and contains two protein binding domains, the beta domain (comprised of residues 63-155 and 193-204) and the alpha domain (residues 155-193) (Fig. 1). The VHL protein (pVHL) utilizes the alpha domain to connect to Elongin B, Elongin C, and CUL2 to form a functional VHL complex, whereas the beta domain is used to recognize substrates (7). The VHL complex regulates the expression of at least 17 cellular proteins, including a key transcription factor known as the hypoxia-inducible factor 1 (HIF). Overabundance of HIF permits the overproduction of growth factors, which stimulate the oncogenic processes that lead to both hereditary and sporadic forms of clear RCC (8,9).
We report the clinical characteristics and functional consequences of a novel VHL, missense, mutation (X214L). Analysis of this mutation supported the Type 2A phenotype in families with a previously unclassifiable molecular subtype of VHL disease.

Patient selection
The proband was referred to the City of Hope (COH) by her surgical oncologist. The proband and her mother were enrolled in a COH IRBapproved Hereditary Cancer Biology Research (HCBR) registry after informed consent. Three additional probands and four at risk familial germline carriers with the same or similar genotypes were identified through a database search by the Molecular Genetics Laboratory at the Children's Hospital of Philadelphia (MGL-CHOP). Additional phenotype data was solicited from the five physicians who referred these patients to the MGL-CHOP, consistent with current HIPAA regulations. One physician responded, his patient   (9)). subsequently provided informed consent and enrolled in the HCBR registry. Medical records and pathologic data were available for the COH cases and one MGL-CHOP case.

DNA analysis
Boston University School of Medicine's laboratory performed CLIA-approved DNA full sequence analysis of the VHL gene for the proband and sitespecific DNA analysis for the proband's mother. Genomic DNA was extracted from peripheral blood lymphocytes using standard procedures. Sequence analysis consisted of polymerase chain reaction (PCR) amplification and automated fluorescence sequencing of all three VHL exons. Multiple ligation probe amplification (MLPA), using the VHL kit from MRC-Holland was also performed. The VHL kit from MRC-Holland detects approximately 97% of all deletion mutations in the VHL gene.

Functional characterization
786-O and RCC-4 VHL null RCCs were cultured in Dulbecco's modified medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. X214L mutant cDNA plasmid was generated from a wild-type pVHL construct by site-directed mutagenesis. A reading frame for 14 extra amino acids was inserted with primers that introduced a 5 BamHI site and a 3 EcoRI site, followed by subcloning into a pBabe retroviral vector that introduces an N-terminal hemagglutinin (HA)-epitope tag. RCCs were infected with retroviruses and selected in 2 μg/ml puromycin for 7-10 days.

Western blot analysis
Twenty micrograms of whole cell extract per lane, measured by Bradford assay, was resolved on 10% or 12% SDS-PAGE gels and transferred to nitrocellulose membrane (Bio-Rad) to detect endogenous HIF2α and JunB. After blocking in TBS with 5% nonfat milk, the membranes were probed with anti-HIF2α monoclonal antibody (UP-11; raised in our laboratory) (10) or anti-JunB monoclonal antibody (C-11; Santa Cruz Biotechnology) or monoclonal anti-HA antibody (HA-11; Santa Cruz). Bound protein was detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and an enhanced chemiluminescence kit (Pierce).

Phenotype
Our proband is a 20-year-old female referred for cancer genetics consultation due to a personal history of two synchronous primary tumors (a second primary pheochromocytoma and a malignant neuroendocrine tumor of the pancreas) and a family history of early-onset pheochromocytomas. A four-generation pedigree was obtained (Fig. 2).
She presented with a less than 6-month history of non-specific abdominal pain, nausea, vomiting, diarrhea, fatigue, and 10-15 pounds of intentional weight loss. Biochemical screening demonstrated an increased normetanephrine level of 487 (normal less than 111); plasma metanephrine level was normal at 47. A diagnostic computed tomography (CT) scan of the abdomen revealed a 3.7 × 4.1 cm ring enhancing mass in the head of the pancreas and an additional mass in the right adrenal gland. No pancreatic or renal cysts were identified. Pathological review confirmed the presence of a 1.6 cm islet cell neuroendocrine carcinoma of the pancreas with nodal metastasis (4 out of 14 lymph nodes were positive) and a benign pheochromocytoma (2 cm in size) associated with medullary hyperplasia of the right adrenal gland. Screening for VHL-associated tumors revealed bilateral retinal HB. Since her surgery 20 months ago, our proband has shown no evidence of recurrent disease.

Genotype
Full sequence analysis on our proband and sitespecific testing of our proband's mother identified a point mutation (X214L) in exon three of the VHL gene (Fig. 1). The X214L (c.641G>T; p.X214LeuextX15) mutation replaces the normal stop codon with a leucine. This run-on mutation, which is predicted to increase the length of the normal pVHL by 14 amino acids, had not been reported in the literature and the clinical significance was unknown.
The MGL-CHOP identified stop codon mutations in three other patients with VHL disease. The MGL-CHOP patient who participated in HCBR registry carries a heterozygous X214C VHL germline mutation (c.642A>T; p.X214CysextX15). This A to T mutation at nucleotide 642 of the VHL gene replaces the stop codon (TGA) in exon 3 with a codon for cysteine (TGT), which is predicted to cause the same elongated protein product seen in our proband and two other MGL-CHOP patients. The specific mutations identified in the other two patients are as follows: X214L The three MGL-CHOP patients who carry stop codon mutations express similar phenotype characteristics as those seen in our X214L family (Table 1). Patient X214C is a 62-year-old male with a history of bilateral pheochromocytomas, multiple central nervous system (CNS) and spinal HB, retinal angiomas, and the synchronous intraoperative finding of a clear RCC with a neuroendocrine tumor of the pancreas. He has undergone bilateral adrenalectomies and several surgical procedures for his spinal tumors. Patient X214L is a 38-year-old female with bilateral retinal angiomas. Patient X214T is a 32-year-old female with pheochromocytoma and pancreatic cysts. The age of onset of disease and family histories are not available for these patients.

Functional data
Prior studies suggest that pVHL mutants linked to familial pheochromocytoma fail to downregulate the c-Jun antagonist JunB, attenuating apoptosis, and setting the stage for neoplastic transformation (10). Using two independent, pVHL null, RCC lines (786-O and RCC-4), we found that the X214L pVHL mutant is associated with abnormal JunB expression, when compared to wildtype pVHL (Fig. 3). The electrophoretic mobility of the exogenous HA-tagged pVHL (X214L) was slower than that of HA-tagged wild-type pVHL, consistent with the presence of 14 additional amino acids. HIF2α induction patterns were assessed after treatment with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase inhibitor, in cell lines expressing wild-type or X214L pVHL.  (21). Amino acids with higher numbers are more hydrophobic and tend to be located on the internal surface of three dimensional protein structure models (22). Hydrophilic amino acids tend to be found on protein surfaces. c Synchronous. d Screen-detected tumor. The X214L mutant pVHL appeared to downregulate HIFα protein expression, in a canonical, hydroxylation-dependent, manner. In contrast, X214L was defective relative to wild-type pVHL with respect to downregulation of JunB. In sum, X214L mutant protein was associated with low HIF2α expression and high JunB expression, a pattern consistent with a low risk of kidney cancer and high risk of pheochromocytoma.

Discussion
This report provides clinical and functional data for a novel VHL variant of uncertain significance (X214L) that is now classifiable as a deleterious mutation. The functional work included in this report supports prior studies that show Jun B dysregulation plays a role in the pathogenesis of Type 2A VHL disease (10). Phenotype expression of VHL is highly variable with marked genotype-phenotype correlation (11)(12)(13)(14). VHL Type 2 families have a high risk of developing pheochromocytoma. The majority of VHL Type 2 mutations occur in the Elongin C binding area (EloC 157-170), which cause only a partial loss of VHL functional ability (15). Type 2 VHL families are divided into subgroups (Types 2A, 2B, and 2C), based on their risk for HB, clear RCC, and characteristics of their germline mutation. Families with Type 2A disease typically carry the more functional 'surface' type of missense mutations. Surface missense mutations are associated with the highest rates of age-related risk for pheochromocytoma (∼60% penetrance by age 60). Type 2A missense mutations are associated with a high risk of HB, but they are associated with a low risk of RCC (16). Interestingly, although Type 2C patients also carry missense types of VHL germline mutations, they do not have an increased risk of developing either HB or RCC. VHL Type 2B families are more similar to Type 1 families, in that they carry more functionally deleterious mutations (partial gene deletions, nonsense mutations, and 'deep' missense mutations) that cause a high risk of renal cancer and a high risk of HB. 'Deep' missense mutations cause more structural damage, because they tend to be hydrophobic residues buried deep within the protein structure (17). Despite the seemingly hydrophobic nature of the VHL X214L missense mutation, this variant has maintained the ability to regulate HIF expression, which is predictive of a low RCC risk. Additional studies are required (e.g. crystal structure of protein, quantitative functional investigations, etc.) to elucidate the unique biological features of this variant.
Pancreatic islet cell tumors are a rare component of the VHL phenotype that has been reported in both Type 2A and Type 2B patients. Less then 12 % of VHL patients express both neuroendocrine tumors of the pancreas and pheochromocytomas (1). Pediatric VHL germline carriers, almost, never express this combination of tumors (18). We contribute an additional case of bilateral pheochromocytoma combined with early-onset neuroendocrine tumor of the pancreas to the literature. The phenotype in this proband and family support findings from a large case series reported by Walther et al (19) who found a statistically significant association (p < 0.025) between mutations at nucleotides 382 through 482 of the VHL gene and the early-onset of pheochromocytomas.
This stop codon mutation predicts the translation of a 'run-on' protein, extending the length of the normal pVHL by 14 amino acids. Similar mutations that create elongated protein products are causative in other inherited disorders. For instance, single base substitutions in the alpha-chain termination codon produce elongated alpha-chain hemoglobin variants that cause an alpha-thalassemia phenotype (20). We identified four families with pheochromocytoma-dominant VHL phenotypes who carry stop codon mutations. Although the elongated pVHL length is the same, the amino acid encoded by the stop codon mutation is different in three of the four families, making shared family origin very unlikely. A search of the VHL mutational databases at Boston University's School of Medicine and the Worldwide VHL Mutations Database revealed the existence of a French family (15) with a germline mutation in the same codon. However, no clinical information (published or anecdotal) about this family could be obtained.
This report expands the knowledge of genotype-phenotype correlations in VHL and reinforces the need to perform functional investigations of variants of uncertain significance in disorders with strong phenotype-genotype correlations.