Retinal Hemangioblastoma in von Hippel-Lindau
Disease: A Clinical and Molecular Study
Hélène Dollfus,1 Pascale Massin,2 Pierre Taupin,3 Catherine Nemeth,4 Sandrine Amara,5
Sophie Giraud,6 Christophe Béroud,7 Pascal Dureau,8 Alain Gaudric,2 Paul Landais,3,9
Stéphane Richard,9,10 for the French VHL Study Group11
PURPOSE. To assess the natural history of retinal manifestations
in von Hippel-Lindau (VHL) disease and to study the genotype–
phenotype correlation.
METHODS. Data concerning 103 patients with VHL retinal man-
ifestations and 108 patients without VHL retinal manifestations
were extracted from the French VHL database. A retrospective
study was performed by questionnaire. Patients were classified
into three visual morbidity groups. Molecular analysis of the
VHL gene was performed in 196 patients.
RESULTS. The mean age of ocular manifestations detection was
24.8 years. In half of the cases, the ocular manifestations
revealed the disease. Half of the cases had bilateral involve-
ment. Visual morbidity was significantly associated with the
retinal hemangioblastoma count but not with other ocular or
general characteristics. One third of the patients were classi-
fied in the worst visual morbidity group at the end of follow-
up. Mutations were detected in 81% of patients with retinal
hemangioblastomas and in 71% of patients without retinal
involvement. Using a Poisson model and a marginal approach,
the number of hemangioblastomas, age-adjusted, was 2.1 times
higher in patients who had a substitution than in patients with
a truncation (95% CI, 1.05– 4.44; P ⬍ 0.05).
CONCLUSIONS. Visual loss remains one of the major complica-
tions of VHL disease, confirming the importance of early oph-
thalmologic screening. Visual morbidity was not related to the
type of extraocular manifestation but appeared to be related to
the type of germline mutation. However, only further genetic
and clinical studies in a larger series of patients will clearly
From the 1Fédération de Génétique, Ophthalmology Department,
Hôpitaux Universitaires de Strasbourg, Strasbourg, France; the 2Oph-
thalmology Department, Hôpital Lariboisière, Paris, France; the 3Bio-
statistics Department, Hôpital Necker-Enfantes Malades, Paris, France;
the 4Ophthalmology Department, Hôpital La Croix Rousse, Lyon,
France; 5the Ophthalmology Department, University Hospital Center,
Clermont-Ferrand, France; the 6Genetics Laboratory, Hôpital Edouard
Herriot, Lyon, France; the 7Institut National pour la Santé et Recherche
Médicale U383, Hôpital Necker-Enfants Malades, Paris, France; the
8
Ophthalmology Department, University Hospital Center, Necker,
Paris, France; and 10Laboratory of Oncogenetics, Ecole Pratique des
Hautes Etudes, Le Kremlin Bicêtre and Department of Nephrology,
Hôpital Necker-Enfants Malades, Paris, France.
9
Contributed equally to the work.
11
Group members are listed in the Appendix.
Supported by grants from the Comité du Cher of the National
League against Cancer.
Submitted for publication September 18, 2001; revised March 22,
2002; accepted April 25, 2002.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Hélène Dollfus, Service de Génétique
Médicale et Service d’Ophtalmologie, Hôpitaux Universitaires de Stras-
bourg, Hôpital de Hautepierre, avenue Molière, 67098 Strasbourg Ce-
dex, France; helene.dollfus@medecine.u-strasbg.fr.
Investigative Ophthalmology & Visual Science, September 2002, Vol. 43, No. 9
Copyright © Association for Research in Vision and Ophthalmology
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determine the genotype–phenotype relationship. (Invest Oph-
thalmol Vis Sci. 2002;43:3067–3074)
V on Hippel-Lindau (VHL) disease (Online Mendelian Inher-
itance in Man [OMIM] 193300) is a dominantly inherited
familial cancer syndrome predisposing to various benign or
malignant tumors: central nervous system (CNS) and ocular
hemangioblastomas, renal cell carcinoma (RCC) and/or renal
cysts, pancreatic tumors and cysts, pheochromocytoma, and
endolymphatic sac tumors.1–7 The birth incidence is estimated
to be 1 in 36,000 to 1 in 53,000.8 –9
Hemangioblastoma is the most emblematic lesion of VHL
disease because of its major clinical implications in the natural
history of the disease.5– 8,10 This benign tumor with a very
singular vascular and cellular (stromal cell) differentiation oc-
curs both in CNS and retina.5– 6,11 The hemangioblastoma may
be a single tumor, and sometimes it is the only manifestation of
the disease, but the tumors are more frequently multiple.
Ocular hemangioblastomas occur in 45% to 60% of patients
with VHL.3–7,12–15 On fundus evaluation, the lesions have an
easily recognizable globular reddish appearance with a dilated
feeding artery and a tortuous draining vein. The size of the
retinal hemangioblastoma may be variable, ranging from a
pinpoint lesion or a discrete vascular tuft to a large globular
lesion (Figs. 1, 2). In spite of its benign nature and classic
slow-growing course, ocular hemangioblastoma may cause
sight-threatening complications and remains a major cause of
visual morbidity or sometimes blindness for patients with VHL
disease. Early detection and treatment can change the visual
prognosis.13,16 –17
Predisposition to VHL results from germline mutations in
the VHL tumor-suppressor gene located on chromosome 3p25-
p26, and development of tumors results from a somatic inac-
tivation of the remaining wild-type VHL allele. Germline muta-
tions in the VHL gene, spanning three exons and encoding a
213-amino-acid protein (pVHL), are now identified in most of
the cases.18 –27
The main functional domains of the pVHL protein have
been recently characterized.28 –34 The major function of the
pVHL protein is the negative regulation of hypoxia-inducible
mRNAs such as the mRNA encoding VEGF by targeting hyp-
oxia-inducible transcription factors (HIFs) for degradation by
the proteasome.35– 42 Indeed, the hallmark of VHL tumors, and
moreover hemangioblastomas, is a high degree of vasculariza-
tion due to overexpression of VEGF.6,43
In spite of these important advances in the understanding of
VHL disease, the overall extremely variable intra- and interfa-
milial expressivity of the disease remains unclear. A genotype–
phenotype correlation has emerged only for the occurrence of
pheochromocytoma, which distinguishes VHL type 1 (without
pheochromocytomas) and VHL type 2 (with high risk for pheo-
chromocytoma).7,19 –23,44 – 45 Type 2 is subdivided in three
subtypes, 2A (with low risk of renal cancer and pancreatic
tumors); 2B (the full multitissue subtype), and 2C (pheochro-
mocytomas only, recently individualized by molecular genet-
ics). Families with VHL type 1 typically have VHL deletions or
3067
 3068 Dollfus et al.
protein truncating mutations, whereas families with type 2
have mainly missense mutations.
The purposes of this study were to analyze the natural
history and the potential genotype–phenotype correlation of
the ocular manifestations in a large series of French VHL pa-
tients.
PATIENTS AND METHODS
The patients were recruited through the French VHL database, which
was created in 1992. During the period of the study (1996 –1999), 610
patients with VHL were registered in the database. Detailed systemic
follow-up data were available for 211 patients from 111 distinct pedi-
grees. The patients were divided into two groups: one group of 103
(48.8%) patients from 60 pedigrees with ocular involvement and a
second group of 108 (51.2%) from 66 pedigrees with no ophthalmic
manifestation reported on the last follow-up. Fifteen families were
involved in both groups.
To be included, the patient had to meet the diagnostic criteria for
VHL defined by the presence of two major manifestations, including at
least one CNS or retinal hemangioblastoma, one major manifestation
and a positive family history, or an isolated clinical feature with muta-
tion in the VHL gene.6 Patients with solitary retinal hemangioblastoma
and no mutation detected in the VHL gene were excluded from the
study.
One hundred fifty-six patients had type 1 VHL, and 55 patients had
type 2 (13 type 2A, 42 type 2B, no type 2C). In the group with ocular
manifestations, 77 patients had type 1 VHL and 26 had type 2 (9 type
2A and 17 type 2B). In the group without ocular involvement, 79
patients had type 1 VHL and 29 had type 2 (4 type 2A, 25 type 2B).
General and Ophthalmological Data Assessment
A questionnaire (available on request) inquiring about the ocular and
general status of the patients was sent to ophthalmologists treating
patients with VHL. Extraocular features were also assessed with the
VHL database.
General Data Evaluation
Personal and familial data were obtained and follow-up duration was
evaluated. Detailed information concerning each extraocular manifes-
tation if present were recorded and included: CNS hemangioblastomas,
renal cell carcinoma, renal cysts, pheochromocytoma, pancreatic
cysts, and/or neuroendocrine tumors, endolymphatic sac tumors.
Ocular Manifestations Assessment
The age of onset and the history of ocular manifestations were re-
corded. Vision at the first and last ophthalmic examination were noted.
Detailed descriptions of the first and last ophthalmic manifestations
were obtained.
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IOVS, September 2002, Vol. 43, No. 9
FIGURE 1. Left: fluorescein angiog-
raphy clearly shows multiple small
retinal hemangioblastomas without
dilation of feeder vessels. Right: fun-
dus photograph of optic disc heman-
gioblastoma that covers the surface
of the disc and expands into the sur-
rounding retina. Fibrosis is also visi-
ble on the surface of the hemangio-
blastoma, which is surrounded by a
serous retinal detachment.
Descriptive Data
The number and location (retinal or papillary) of the retinal heman-
gioblastomas were recorded for each eye at initial and final examina-
tion. Presence or absence of complications due to the retinal heman-
gioblastomas were noted. The following items were considered:
macular exudation, extramacular exudation, retinal traction, retinal
detachment treated successfully with surgery, retinal detachment with
unsuccessful surgical treatment, optic disc or hemangioblastoma neo-
vascularization, neovascular glaucoma, vitreous hemorrhage, phthisis,
enucleation, and any other relevant ocular feature. Photographs and
fluorescein angiograms were obtained if possible.
Impact on Visual Function
Visual acuity was measured with the Monoyer decimal charts. The
results were converted to the Snellen 20-ft notation. The patients were
classified according to visual morbidity in three categories: group 1
with normal vision (both eyes ⬎20/40), group 2 with moderate vision
loss defined by vision in at least one eye inferior to 20/40, and group
3 with severe visual loss defined by the vision in one eye inferior to
20/200.15
DNA Mutation Analysis
This study was approved by the Ethics Committee at Le Kremlin-
Bicêtre University Hospital (France) and was conducted adhered to the
tenets of the Declaration of Helsinki. Blood samples were collected
after written consent was received from each patient and processed for
DNA isolation using standard methods. To detect the presence of a
mutation, PCR and single-strand conformation polymorphism (SSCP)
were used systematically. Briefly, we used six sets of primers, de-
scribed elsewhere.46 If no abnormal migration pattern was detected,
we performed direct sequencing. Finally, if both PCR-SSCP and direct
sequencing produced normal findings, a Southern blot analysis was
performed to detect large rearrangements.
PCR-SSCP Analyses. PCR and SSCP analyses were performed
according to the protocol of Gallou et al.46 PCR mixtures (25 ␮L)
contained 20 ng of genomic DNA, 1⫻ PCR buffer, 0.8 ␮M of each PCR
primer, 200 ␮M of each dNTP, and 0.5 U Taq DNA polymerase
(Invitrogen Life Technologies, Cergy Pontoise, France). The reaction
mixtures underwent the following incubations in a thermocycler
(Gene Amp 9600; Perkin-Elmer, Wellesley, MA): 94°C for 5 minutes; 30
cycles of 94°C for 15 seconds and 62°C to 70°C for 30 seconds
(specific for each set of primers); and a final extension at 72°C for 5
minutes. SSCP analysis was performed as previously described.46 Four
microliters of each PCR product containing 0.5 ␮Ci [33P]-dATP was
denatured for 10 minutes at 95°C in 4 ␮L gel-loading buffer and then
chilled on ice. Samples were electrophoresed at 10 W for 10 to 16
hours at 20°C on mutation detection gel (Hydrolink Mutation Detec-
tion Enhanced gel; Bioprobe Systems SA, Montreuil, France).
 IOVS, September 2002, Vol. 43, No. 9 Retinal Hemangioblastoma in von Hippel-Lindau Disease 3069

FIGURE 2. Top left: typical midsized

retinal hemangioblastoma with two
arterial feeder vessels and one dilated
vein. Top right: early phase of fluo-
rescein angiography. Middle left: ret-
inal hemangioblastoma with dilated
afferent and efferent vessels, compli-
cated by hard exudates that imperil
the macula. Middle right: early phase
of fluorescein angiography. Bottom
left: large retinal hemangioblastoma
with major vascular dilation. Fundus
photograph shows preretinal fibrosis
stretching tautly from the surface of
the hemangioblastoma to the optic
disc. Bottom right: fluorescein an-
giography shows dilation of efferent
vessels, rapid staining of the angi-
oma, and the presence of new ves-
sels on the optic disc.

Sequence Analyses. The PCR products were extracted with a
purification kit (Wizard Prep; Promega, Madison, WI) purification kit.
Cycle sequencing was performed on both strands either with a se-
quencing kit (Pharmacia France, St. Quentin Yvelines, France) or with
a sequencing kit (Thermosequenase; Amersham, Succursale, France)
on an automated sequencer (ALF; Pharmacia).
Southern Blot Analysis and Hybridization
DNA samples (6 ␮g) were digested by the restriction enzymes AseI and
EcoRI and run using standard procedures.46 The membranes were
hybridized with the g7 clone labeled with a random priming procedure
for Southern blot analysis (Random Primed DNA labeling kit; Roche
Molecular Biochemicals, Meylan, France).17 Washes were performed at
42°C for 10 minutes in 2⫻ SSC and 0.1% SDS and two times for 5
minutes each at 65°C in 2⫻ SSC and 0.1% SDS. Membranes were
exposed to film for at least 12 hours with intensifying screens at – 80°C.
Statistical Methods
The study was retrospective. For univariate analyses, differences be-
tween groups were analyzed with one-way analysis of variance for
continuous variables.47 Comparisons between categorical covariates
were performed by ␹2 test or the Fisher test with contingency tables.
All tests were two-tailed, and P ⱕ 0.05 was considered to indicate
statistical significance.

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 3070 Dollfus et al.
FIGURE 3. Distribution of patients according to age at first detection
of retinal hemangioblastoma.
A multivariate logistic regression model was used to estimate the
adjusted significance of multiple variables for association with the
incidence of retinal hemangioblastomas: age, sex, CNS hemangioblas-
tomas, renal cell carcinoma, renal cysts, pheochromocytoma, pancre-
atic tumors and cysts, and endolymphatic sac tumors.
The variables were entered in a mixed step-wise fashion into
logistic regression analyses and tested for an independent effect. We
did not add interaction terms. The limit for entering or removing
variables in the logistic regression models was a P ⱕ 0.10. The com-
parison of models was based on the likelihood ratio statistic, which has
an asymptotic ␹2 distribution.
A Poisson distribution was used to model the number of heman-
gioblastomas. A marginal model taking into account the family effect
and age-adjusted was used to compare the number of hemangioblas-
tomas in patients who had a substitution or a truncation.
Statistical analyses were performed on computer (S-Plus ver. 4.5;
Mathsoft, Cambridge, MA; and SAS ver. 8.2; SAS Institute, Cary, NC).
RESULTS
Patients’ General Data
The mean duration of follow-up was 8.4 years (range, 1–39) for
the group of patients with ocular manifestations and was 7.6
years (range, 1– 42) for the group of patients without ocular
involvement. Seventy-six patients of the ocular group had a
positive familial history, and 27 patients were cryptic (isolated
cases without family history). For the group without ocular
manifestations, 92 patients had a positive family history, and 16
were cryptic.
Natural History
The mean age at the moment of VHL diagnosis was 24.8 years
(range, 6 – 60) in the group of patients with ocular manifesta-
tions and was 31 years (range, 10 – 41) in the group of patients
without ocular manifestations. The mean age of patients with
retinal manifestations was significantly lower than that of pa-
tients without ocular manifestations. This difference created
difficulties in the matching of the two groups of patients. A
similar difference was present in a British series.14 –15
The mean age at diagnosis of ocular manifestations was 24.4
years (range, 6 –51). Distribution of patients according to age at
detection of retinal hemangioblastoma is shown in Figure 3.
Ocular lesions revealed the VHL condition in 50% of the cases
in the first group of patients.
The presence of retinal hemangioblastomas was discovered
in different circumstances: because of visual symptoms in 40%
of the patients, in the context of an initial VHL evaluation in
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IOVS, September 2002, Vol. 43, No. 9
19% of the patients, during systematic follow-up VHL evalua-
tion in 16% of the patients, and because of a positive family
history or because of the detection of a VHL mutation in an
asymptomatic VHL carrier in 25% of the patients.
Hemangioblastoma Count
The mean number of hemangioblastomas per gene carrier with
ocular manifestation was 1.35 at initial evaluation (range, 1–20)
and 2.97 (range, 1–20) at final evaluation. During the time of
follow-up, the mean number of new hemangioblastomas per
patient (of the group with ocular manifestation) per year was
0.36. The hemangioblastoma count increased with the dura-
tion of follow-up, but this increase varied according to age
group. Indeed, the number of new hemangioblastomas per
year was higher in the 10- to 20-year-old age group than in the
20- to 30-year-old age group, in which the number was higher
than in the 30- to 50-year-old group. This estimation of the rate
of new lesions appearing during 1 year in one gene carrier is
probably imperfect, and the so-called velocity may vary con-
siderably from one patient to the other.
Optic disc hemangioblastomas were diagnosed in 11% of
the eyes at the first report (5% of the eyes had optic disc and
retinal hemangioblastomas). At final report, 15% of the eyes
had optic disc hemangioblastomas (11% of the eyes had optic
disc and retinal hemangioblastomas). At initial report, 72% of
the patients had retinal hemangioblastomas in one eye and 28%
had them in both eyes. At final report, 51% of the patients had
retinal hemangioblastomas in one eye only and 49% had bilat-
eral involvement.
Evaluation of Ocular Complications
At initial report, macular exudation was present in 15% of the
eyes, and extramacular exudation was present in 22%. At final
report, macular exudation was present in 9% of the eyes and
extramacular exudation in 12%.
Tractional retinal detachment was reported in 22% of the
eyes at initial report. At final report, 11.5% of the eyes had a
successfully treated retinal detachment, whereas 17% of the
eyes had a retinal detachment for which surgery had been
ineffective.
One eye was reported as having new vessels (on a periph-
eral hemangioblastoma). No optic disc neovascularization was
reported. Ten (5%) eyes had neovascular glaucoma and six
(3%) had to be enucleated because of pain and/or phthisis
bulbi.
Evaluation of Visual Impairment
Visual morbidity was evaluated by classifying the patients into
three groups according to their visual acuity (Table 1). On
initial examination 70% of the patients were classified into
group 1, 9% into group 2, and 21% into group 3. On final
examination 64% of the patients were classified into group 1,
6% into group 2, and 30% into group 3. No significant relation
was found between final visual morbidity and the duration of
TABLE 1. Visual Morbidity at Initial Evaluation and Final Evaluation
Group 2 Group 3
(At Least (Vision in
Group 1 One Eye One Eye
(Normal Vision) <20/40) <20/200)
Initial evaluation 70 9 21
Final evaluation 64 6 30
Average follow-up was 8 years. Data are the percentage of total
patients classified in each group.
 IOVS, September 2002, Vol. 43, No. 9
TABLE 2. Incidence of Other VHL Manifestations in Patients of the Study
CNS hemangioblastoma
Renal cell carcinoma
Pheochromocytoma
Pancreatic neuroendocrine tumor
Data are number of patients with each tumor type with percentage of total patients in each category
in parentheses.
follow-up, the age at diagnosis, initial papillary involvement,
and ocular involvement as a first sign of VHL disease. On the
contrary, a significant association was found between visual
morbidity and the count of hemangioblastomas (P ⬍ 0.001):
Visual morbidity increased with the hemangioblastoma count.
Patients with papillary hemangioblastoma did not differ in
visual morbidity from those without papillary hemangioblas-
toma.
When the group of patients with retinal hemangioblastomas
and the group of patients without ocular manifestations were
compared, no significant association was found concerning the
occurrence of retinal hemangioblastomas and central nervous
system hemangioblastomas, renal cell carcinoma, pheochro-
mocytoma, or pancreatic lesions (Table 2).
Genotype Analysis
In the group with ocular manifestations, 91 (88%) patients
were screened for mutation in the VHL gene, and 74 mutations
were detected (mutation detection rate: 81%). In the group
without ocular manifestations, 105 (97%) patients were
screened for mutations in the VHL gene, and 75 mutations
were detected (mutation detection rate: 71%). Germline muta-
tions in the two groups are detailed in Table 3, and their
locations are represented in Figure 4.
Briefly, mutations in patients with retinal hemangioblasto-
mas include missense mutations in 45 patients; nonsense mu-
tations, frameshifts, or large deletions in 28 patients; and a
splice site mutation in 1 patient. In the group of patients
without retinal manifestations, there were missense mutations
in 44 patients and nonsense mutations, frameshifts, or large
deletions in 31 patients.
No significant differences were established comparing the
location of the mutation (exon or main functional domains of
pVHL). However, the type of mutation (leading to a truncated
protein or to an amino acid substitution) between the two
groups of patients showed a significant association with the
number of hemangioblastomas. The count of hemangioblas-
toma was modeled with a Poisson model. The analysis, using a
marginal model, took into account the familial structure and
was age adjusted. The number of hemangioblastomas appeared
2.1-fold higher in patients who had a substitution when com-
pared with patients with a truncated protein (95% CI, 1.05–
4.44; P ⬍ 0.05).
In the group with ocular manifestations, no significant re-
lation was found between any specific genotype and the ve-
locity of new retinal lesions or the retinal hemangioblastoma
count, and no significant association found with the visual
morbidity, as defined earlier.
DISCUSSION
Retinal hemangioblastoma is one of the most frequent tumors
occurring during the course of VHL disease and can be respon-
sible for significant visual impairment. This study was designed
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Retinal Hemangioblastoma in von Hippel-Lindau Disease 3071
Patients with Retinal Patients without Retinal
Hemangioblastomas Hemangioblastomas
(n ⴝ 103) (n ⴝ 108)
77 (75%) 83 (77%)
53 (51%) 46 (43%)
21 (20%) 23 (21%)
13 (16%) 17 (16%)
to contribute to defining the natural history, the visual morbid-
ity, and genotype–phenotype correlation in a French popula-
tion with VHL disease, comparing patients with and without
ocular manifestations. As opposed to other studies, this study
took into account the duration of follow-up (on average, 8
years) for the two groups of patients.14 –15,48 However patients
were managed in different centers with different observers,
and therefore the variability of the care provided in the centers
and by the ophthalmologists weakens the study.
The impact of the ocular evaluation in VHL is considerable,
because the first manifestation of VHL was ophthalmic in
50% of the patients. The ocular manifestations are known to
occur early in the course of the disease, as confirmed by our
series.3– 4,7,14 –16,48 Indeed, persons between 15 and 25 years
of are in a critical age group for the development of retinal
hemangioblastomas. Patients with VHL in this age group war-
rant careful ophthalmic follow-up, keeping in mind the
younger (6 years old) and older patients (60 years old) of this
series diagnosed with retinal hemangioblastomas. The fol-
low-up guidelines for the care of retinal hemangioblastomas in
France advocates yearly ophthalmic examination from the age
of 5 years. Between the age of 15 and 30 years, a funduscopy
is recommended twice a year. After 30 years of age, a yearly
examination is recommended. The rate of new hemangioblas-
tomas in elderly patients may be higher than expected from the
data available to date because of the increasing life expectancy
due to considerable improvement in the clinical management
of VHL.
Visual symptoms, usually related to long-standing or active
lesions, revealed ocular involvement in 40% of the patients.
This result in our series shows the necessity of improving the
systematic early detection of lesions.
Eleven percent of the eyes at initial examination and 15% of
the eyes at final examination exhibited papillary hemangioblas-
tomas. Optic disc hemangioblastoma occurred in 8.3% of the
eyes in a British series.15 In our series, no hemangioblastoma
was present at the posterior pole other than at the optic disc.
Macular exudation and retinal detachment are the major
complications induced by retinal hemangioblastomas. During
follow-up, a decrease of the number of patients with exudation
was noticed. There are two explanations for this: efficient
treatment for some patients and absent final evaluation of the
exudation in patients with advanced retinal lesions. At final
evaluation, 17% of the eyes had a retinal detachment for which
surgery was ineffective, and 3% of the eyes had to be enucle-
ated.
At final examination, 30% of the patients were classified in
the severe visual loss group, compared with 21% at the begin-
ning of follow-up. In this series, the risk of visual impairment
increased with the hemangioblastoma count but not with the
age at discovery or with the occurrence of visual symptoms.
For these two last points, the results herein are not in accor-
dance with those in a previous series.15
 3072 Dollfus et al. IOVS, September 2002, Vol. 43, No. 9

TABLE 3. Details of VHL Germline Mutations in Patients in the Study

Patients Patients
Wild-Type with without
Wild-Type Amino Protein Retinal Retinal
Mutational Event Type Localization Codon Codon Acid Consequence Hb (n) Hb (n)

136G3T Ts Exon1 46 GAG E E46X 1 0

154G3A Ts Exon1 52 GAG E E52K 0 1
161insG Fs Exon1 54 ATG M Stop131/no initiation 0 3
179delG Fs Exon1 60 CGG R Stop66 0 1
193T3C Ts Exon1 65 TCG S S65P 0 2
197del24 Fs Exon1 66 GTG V Frame shift 1 0
211insT Fs Exon1 71 CCC R Stop131 0 1
217C3T Ts Exon1 73 CAG Q Q73X 4 1
226–228delTTC In Exon1 76 TTC F In-frame deletion 0 2
233A3G Ts Exon1 78 AAT N N78S 1 1
238A3C Tv Exon1 80 AGT S S80R 1 0
239G3A Ts Exon1 80 AGT S S80N 2 0
245G3C Tv Exon1 82 CGC R R82P 1 2
256C3T Ts Exon1 86 CCC P P86S 1 2
263G3C Tv Exon1 88 TTC F F91L 1 0
286C3T Ts Exon1 96 CAG Q Q96X 1 2
311G3C Tv Exon1 104 GGC G G104A 1 0
313A3C Tv Exon1 105 ACG T T105P 5 4
320G3A Ts Exon1 107 CGC R Stop131 0 1
322insTG Fs Exon1 108 CGC R Stop131 0 1
331A3T Tv Exon1 111 AGC S S111C 3 0
337C3T Ts Exon1 113 CGA R R113X 2 0
340G3C Tv Exon1 114 GGT G G114R 1 0
IVS1 ⫹ 1G3A Ts IVS1 Donor splice site — — Splice 1 0
343C3T Ts Exon2 115 CAC H H115Y 1 0
345C3G Tv Exon2 115 CAC H H115Q 0 1
351G3T Tv Exon2 117 TGG W W117C 4 1
353T3C Ts Exon2 118 CTC L L118P 1 0
386insCT Fs Exon2 129 CTG L Stop131 0 1
388G3C Tv Exon2 130 GTT V V130L 2 1
395A3C Tv Exon2 132 CAA Q Q132P 0 1
407T3C Ts Exon2 136 TTT F F136S 2 0
417–418delTC Fs Exon2 139 TCT S Stop142 0 1
439insT Fs Exon2 147 ATT I Stop173 0 1
462A3C Tv Exon2 154 CCA P P154P 1 0
463G3C Tv Exon3 155 GTG V V155L 1 1
466T3G Tv Exon3 156 TAT Y Y156D 0 1
467A3G Ts Exon3 156 TAT Y Y156C 1 2
468T3G Ts Exon3 156 TAT Y Y156X 0 3
473insT Fs Exon3 158 CTG L Stop173 1 0
473insTTT Fs Exon3 158 CTG L Stop173 1 1
473T3C Ts Exon3 158 CTG L L158P 1 0
481C3T Ts Exon3 161 CGA R R161X 3 5
482G3A Ts Exon3 161 CGA R R161Q 3 0
486C3G Tv Exon3 162 TGC C C162W 0 4
496G3T Tv Exon3 166 GTC V V166F 2 0
499C3T Ts Exon3 167 CGG R R167W 5 10
500G3A Ts Exon3 167 CGG R R167Q 3 7
525C3A Tv Exon3 175 TAC Y Y175X 0 1
525C3G Tv Exon3 175 TAC Y Y175X 3 4
563T3C Ts Exon3 188 CTG L L188P 1 0
571delC Fs Exon3 191 CAC H Stop201 1 0
604–605delAC Fs Exon3 202 ACA T No stop 0 1
612–613delGC Fs Exon3 204 GAG E No stop 0 1
Large rearrangement — — — — — — 9 3

Nucleotides are numbered according to the international VHL data base.26 Hb, hemangioblastoma.

This study did not demonstrate a significant relation be-
tween retinal hemangioblastomas and other systemic VHL
manifestation. Moreover, the incidence of retinal hemangio-
blastomas was not increased in the presence of CNS heman-
gioblastomas, which share the same histologic features. These
results contradict those in a previous study in which patients
with ocular hemangioblastoma had a significantly increased
incidence of cerebellar hemangioblastoma and renal cell carci-
noma.14 –15 In this study, we could not show that the risk of
development of a renal cell carcinoma or CNS hemangioblas-
toma was different between the two groups of patients with or
without ocular involvement. However, our logistic approach
did not take into account the date of onset of each extraocular
lesion. The clinical follow-up of extraocular manifestations of
the disease should be identical in the presence or absence of
retinal hemangioblastoma.
A genotype–phenotype correlation in VHL has emerged
with confirmation of the clinical pheochromocytoma-based

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 IOVS, September 2002, Vol. 43, No. 9 Retinal Hemangioblastoma in von Hippel-Lindau Disease
FIGURE 4. Distribution of VHL gene point mutations in patients in the
study. The main functional domains are indicated. ␤, ␤ domain; ␣, ␣
domain; EloC, elongin C; HIF, hypoxia-inducible factor-␣; 167, hot-spot
of mutation in VHL disease.
distinction.7,20 –22,27,44 – 45,49 –50 Two types of VHL are distin-
guished according to the presence or absence of pheochromo-
cytoma, with close correlations with the type of mutation. In
type 1, patients can have all manifestations except pheochro-
mocytomas. In type 2, on the contrary, pheochromocytoma is
the main characteristic of the disease. A low risk of renal cell
carcinoma and neuroendocrine pancreatic tumor is associated
with type 2A and a high risk with type 2B. In VHL type 1
(without pheochromocytoma), most of the mutations are de-
letions, insertions, nonsense, and frameshift mutations, result-
ing in a truncated protein. On the contrary, in VHL type 2 (with
pheochromocytoma), most mutations are missense. In type 2B,
mutations affect the critical contact region between pVHL and
elongin C, with a hot spot at codon 167. In type 2A (with
pheochromocytoma and low-risk of renal cell carcinoma) a
specific mutation at codon 98 in several families from distinct
countries was identified, and haplotype analysis showed that it
was caused by a founder effect occurring in the Black Forest in
the 16th century.44 In the more recently individualized type
2C, the disease is characterized by the familial occurrence of
isolated pheochromocytomas only.7,45
In this series, we did not find any clustering of mutations in
the patients with retinal hemangioblastomas and in particular
at the functional binding site of the VHL protein with HIF.
However, in this study, we found a significant association
between the number of hemangioblastomas and the type of
mutation. The number of hemangioblastomas, age and family
adjusted, was 2.1 times higher in patients with a substitution,
when compared with patients with a truncated protein (95%
CI: 1.05– 4.44; P ⬍ 0.05). This finding must be interpreted with
caution, because only by thorough and parallel genetic and
clinical studies of a larger series of patients will a genotype–
phenotype relationship in VHL disease be more clearly deter-
mined. As do other investigators, we believe that patients
should be screened on a regular basis for retinal lesions, what-
ever mutation they carry (except for the very rare type 2C).
Variable expressivity, a hallmark of inherited cancer syn-
dromes, remains poorly understood. Modifier factors, genetic
and/or environmental, are thought to influence the expressiv-
ity of familial cancer syndromes. Evidence has been provided
that unknown modifier genes and environmental influences
play an additional role in the clinical expression of retinal
hemangioblastomas.14 The identification of such factors re-
mains to be achieved and necessitates analyses of a very large
series of patients in an international collaboration.
Downloaded from iovs.arvojournals.org on 03/17/2020
3073
Acknowledgments
The authors thank Guy Allègre and Rachel Messaoudi for technical
assistance.
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APPENDIX
Participating Ophthalmologists and
Their Locations
Jean-Pierre Agez (Enghien-Les-Bains); Claire Audouin-Barthelat
(Ambert); Franck Bacin (Clermont Ferrand); Martine Banche-
reau (Le Mans); Dominique Baudet (Rennes); Franck Becquet
(Nantes); André Benko (Chezelles); Jean-Pierre Biestro
(Dieppe); Bernard Bievellez (Lyon); Mireille Bonnet (Lyon);
Mireille Bouchet (Mont De Marsan); Gilles Chaine (Bobigny);
Isabelle Cochereau-Massin (Paris); Dominique Commeau
(Cormeilles En Parisis); George Constantinides (Lille); Philipe
Cordier (Nevers); Gabriel Coscas (Créteil); Christian Creton
(Metz); Bernard Deroche (Angers); Pierre Devin (Marq-En
Baroeul); Curan Dominique (Creil); Jean-Louis Dufier (Paris);
Claire Fagart (Le Mans); Jacques Flament (Strasbourg); Jean-Luc
Franco (Montluçon); Jean-Pierre Francois (Lille); Marie-Paule
Francois (Joeuf); Chantal Gaianopoulo (Aulnay-Sous-Bois); Jean
Didier Grange (Lyon); Pascale Houtteville (Caen); Jean Ivanez
(Douai); Elisabeth Joubaud-Nenert (Guérande); Marie Laval
(Brive); Martine Logeais (Rennes); Raymond Louly (Cambrai);
Etienne Magniere (Montluçon); Jean Maugery (Saint-Etienne);
André Mathis (Toulouse); Claire Monin (Paris); Dominique
Mouilleau (Le Mans); Michel Paques (Paris); Michel Pomes
(Maisons-Alfort); Antoine Raspiller (Nancy); Philippe Razemon
(Lille); Anne Robert (Brest); Dominique Salame (Paray-Le-Mo-
nial); Annie Salvanet (Maisons-Alfort); Anny Segal (Reims);
Gisèle Soubrane (Créteil); Jean-Pierre Turcat (Chambéry); Jean-
Pierre Vermelle (Liévin).