Abstract
Purpose.:
von Hippel-Lindau (VHL) disease is a dominantly inherited, multisystemic tumor syndrome caused by mutations in the VHL gene. This study was conducted to establish genotype–phenotype correlations between the positions of disease-causing missense mutations and the ocular phenotypes of VHL disease.
Methods.:
Participants with clinically defined VHL disease and documented germline missense mutations in the VHL gene were identified in a cross-sectional study (n = 412). Statistical analysis was used to correlate the position of the missense mutation in either the α- or β-domain of the VHL protein with the ocular disease phenotype.
Results.:
Missense mutations among study participants were located in 47 of the 213 possible codons in the VHL gene. Almost all mutations (98.5%) were located in one of two structural domains of the VHL protein: the α- and β-domains. α-Domain mutations were significantly associated with a higher prevalence of retinal capillary hemangioblastomas (RCHs) compared with the β-domain mutations (P = 0.016). Among patients with RCHs, the prevalence of the lesions in the juxtapapillary position was also significantly higher in patients with α-domain mutations (P = 0.0017). Conversely, β-domain mutations correlated with a higher prevalence of peripherally located RCHs (P = 0.0104).
Conclusions.:
The location of missense mutations in the VHL gene correlates significantly with the prevalence and phenotype of ocular disease, and as such, influences the risk of visual loss in affected patients. These genotype–phenotype correlations can assist in the prognostic counseling and follow-up of VHL patients and may provide a basis for molecular inferences on ocular VHL disease pathogenesis.
Von Hippel-Lindau (VHL) disease (Online Mendelian Inheritance in Man 193300;
http://www.ncbi.nlm.nih.gov/Omim/ provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD) is a dominantly inherited cancer syndrome with multisystemic involvement, caused by mutations in the VHL tumor-suppressor gene.
1 In the eye, VHL disease manifests as benign retinal capillary hemangioblastomas (RCHs), a pink, globular, vascular lesion, located typically in the peripheral retina or in the juxtapapillary area.
2 RCH lesions may, through exudative and/or tractional effects, result in vision loss and disruption of ocular structures,
2–4 constituting a significant part of overall VHL disease–associated morbidity.
5
As described by the two-hit hypothesis of Knudson,
6 affected patients inherit one mutated copy of the
VHL gene from an affected parent through the germline. Later in life, the other normal copy of the
VHL gene undergoes somatic mutation in susceptible tissues (i.e., the second hit), initiating local tumorigenesis. The VHL protein is ubiquitously expressed,
7 and its molecular function is primarily associated with the formation of a ubiquitin ligase complex with other participating proteins (elongin B, elongin C, cullin 2, and Rbx1) that subsequently bind and direct the degradation of the transcription factor hypoxia inducible factor (HIF).
8 The loss of
VHL function in mutated cells is thus thought to result in the dysregulated accumulation of HIF, which then directs the excessive transcription of downstream genes, including angiogenic growth factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).
7,9,10 In the retina, elevations of these angiogenic growth factors are thought to promote the RCH formation and growth observed in ocular VHL disease.
11,12
With the identification of the
VHL gene,
1 genetic diagnostic tests that detect germline mutations in the gene have been made available,
13 enabling early disease diagnosis, improved tumor surveillance, and a reduction of disease morbidity.
14,15 With this technology, a variety of mutation types have been since been characterized
16 and curated in databases (
http://www.umd.be/).
17 With this information, genotype–phenotype correlations have also been established,
18 aiding patient counseling concerning disease prognosis, as well as furthering a molecular understanding of the function of the VHL protein.
19
With respect to ocular VHL disease, in a large group of genotyped participants (
n = 873) with clinically defined VHL disease, in earlier work we established genotype–phenotype correlations between the nature of the mutation (amino acid substitutions, protein-truncating mutations, and complete deletions of VHL protein) and the prevalence of ocular disease and visual function.
20 However, the impact of missense mutations in different parts of the
VHL gene on ocular phenotype has not been analyzed. In the present study, we examined a subset of 412 participants with clinical VHL disease, in whom the germline
VHL mutation consisted of a missense mutation resulting in a single amino acid substitution in the VHL protein. In this study, we established correlations between the position of the missense mutation with the ocular phenotype, to examine how point mutations arising from different VHL protein domains may influence the nature of retinal angiomatosis. We analyzed the position of missense mutations according to their location in two main functional domains: the α-domain, which organizes the ubiquitin ligase complex by interacting with elongin B/C, Cul2 and Rbx1, and the β-domain, which binds the substrate HIF.
21–24 We examined correlations between the domain position of missense mutations with the (1) prevalence of RCHs among VHL patients, (2) the ocular phenotype of RCHs, including RCH position, number, and severity, and (3) visual function. These correlations can assist in understanding how VHL gene mutations in particular affect the formation and development of ocular VHL disease and provide prognostic information for counseling and monitoring ocular disease in VHL patients.
Participants in this study were enrolled in a protocol studying VHL disease from October 1988 to August 2005 at the National Cancer Institute (Bethesda, MD). The study adhered to the tenets of the Declaration of Helsinki and was approved by a local institutional review board (IRB). Informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study. The participants were screened for evidence of clinically defined systemic VHL disease at a single center. Evaluations included history and physical examinations by a multidisciplinary team, laboratory evaluations, and radiographic imaging (computed tomography or magnetic resonance imaging) of the abdomen, pelvis, brain, and spine. All participants also underwent ophthalmic examination with slit lamp biomicroscopy, indirect funduscopy, and, if indicated, fluorescein angiography. A total of 890 participants who met the clinical diagnostic criteria for VHL disease were examined and analyzed. The participants were followed up over time, and treatments for ocular VHL disease were provided as clinically indicated. Analysis of present participant information was performed in a cross-sectional design, with each participant's most recent visit designated as the study visit.
Participants were evaluated with a full ophthalmic examination of both eyes, when available, including the measurement of best corrected visual acuity with the Early Treatment of Diabetic Retinopathy Study visual acuity chart, intraocular pressure measurement, slit lamp biomicroscopy, and indirect funduscopy. Each eye of every participant was evaluated in the study and scored for the presence of ocular involvement by VHL disease. Eyes with ocular VHL disease were scored for presence of severe tumor involvement, defined as causative of enucleation or phthisical and prephthisical changes that precluded an adequate view into the posterior pole. In less severely affected eyes in which the posterior pole could be visualized on fundus examination, the extent of retinal involvement (scored as the number of quadrants of the retina affected by ocular angiomatosis) was noted. In addition, the number and location (juxtapapillary or peripheral) of individual RCHs were also recorded. RCHs were classified as juxtapapillary RCHs if they were located on or adjacent to the optic nerve and as peripheral RCHs if they were located peripheral to the vascular arcades and twice the fovea-to-disc distance from the optic nerve.
Participant screening procedures involving systemic evaluations for VHL disease resulted in the identification and enrollment of 890 participants with clinically defined disease. Genotype information was obtained in 873 (98%). Of these, 412 (47.2%), originating from 135 separate family pedigrees, were found to have missense mutations that resulted in a single-residue substitution in the amino acid sequence of the VHL gene. The demographic features of these 412 participants with missense mutations were as follows: mean age ± SD, 37.1 ± 15.6 years (range, 4.2–84.3); sex, 186 male and 226 female (male-female ratio 1:1.22); and self-reported race/ethnicity, 381 (93%) white, 18 (4%) Hispanic, 10 (2%) black, and 3 (1%) Asian.
Relationship between Location of Missense Mutations in the α-Domain versus the β-Domain and the Prevalence of Ocular VHL Disease
Relationship between Location of Missense Mutations in the α-Domain versus the β-Domain and VHL Ocular Phenotype
Relationship between Position of Missense Mutations in the α-Domain versus the β-Domain and Visual Acuity
Effect of Location of Missense Mutations in Specific Functional Subdomains on Ocular VHL Disease