Von Hippel Lindau Disease in Kerala - All India Ophthalmological ...

180 AIOC 2010 PROCEEDINGS
Von Hippel Lindau Disease in Kerala: A Clinical and Genetic
Study
Dr. Gopal S. Pillai, Dr. Natasha Radhakrishnan, Dr. Ashok Pillai
(Presenting Author: Dr. Gopal S. Pillai)
The Von Hippel-Lindau disease (VHL) is an
inherited disorder involving a variety of
malignant and benign neoplasms of the retina,
central nervous system, and abdominopelvic
viscera. Most frequent patterns described in
literature involve retinal angiomatosis, cerebellar
and spinal hemangioblastomas in combination
with renal cell carcinoma, pheochromocytoma,
and pancreatic tumours.
Its inheritance follows a Mendelian autosomal
dominant pattern. The primary management of
most symptomatic lesions of this complex,
whether benign or malignant, is essentially
surgical. Hence, timely recognition of the
syndromic diagnosis is the secret to achieving the
best long-term clinical outcome. For example,
renal cell carcinoma is a common cause of
untimely death in VHL patients.
Regular abdominal screening of the patient
identified with VHL, allows for early detection of
renal cell carcinoma and total excision with a
partial nephrectomy, thus preserving functional
kidney for the patient. Similarly, through
periodic CNS imaging, early detection of small
cerebellar and brainstem hemangioblastomas is
possible, which can be managed noninvasively
today with stereoradiosurgery. This principle of
constant surveillance and early detection applies
to all tumours involved in this disease complex,
allowing minimization of the neurological and
systemic morbidity.
Western literature sites an average delay in
recognition of the VHL syndrome of 4-5 years
when molecular methods are not used, during
which time the disease may progress to more
advanced stages. To our knowledge, less than
five publications exist describing the VHL
complex in Indian patients, the majority being
only isolate case reports. To date, no studies have
been done addressing the genetic aberrations in
our population.
A gene encoding for a tumour suppressor protein
factor, the loss of which is responsible for VHL
disease, was first identified in 1993 and mapped
to human chromosome 3p25. This gene encodes
for three exons leading to the production of a 213
amino acid protein known as hypoxia inducible
factor (HIF). This protein factor and its metabolic
pathways have subsequently been studied
extensively and found to be an important
mediator in protection against endogenous
oxidant mediated DNA injury.
The role of VHL gene protein product has being
identified in numerous tissues and mutations of
the gene have been described in other neoplasms
outside of the VHL complex. Similar VHL gene
mutations have been found in a high percentage
(60-80%) of tumour DNA from
hemangioblastomas, renal cell carcinoma and
other tumours of the complex occuring
sporadically. The role of VHL gene aberrations in
the development of the VHL disease is described
based on the ‘two-hit hypothesis’. According to
this, germline mutations imply that each cell will
carry one altered copy (the first hit) due to the
inherited germline mutation and one normal
copy. During the individual’s lifetime, when the
second mutation or ‘hit’ in the only remaining
functional copy occurs, the gene function is lost
in that cell. The subsequent lack of cellular
mechanisms vital to protection from oxidant
stresses leads to eventual oncogenesis.
Summarise described mutation patterns in other
populations.
To study the phenotypic and genotypic features
of VHL in Indian population and to evaluate
whether genotypes can be correlated with
phenotypes as in the organ involved, in the
families at risk.
Goal of the study: As a candidate disease with
multiple organ involvement, we tried to study
VHL as a prototype and see if the genotypic and
phenotypic manifestations match among
families. If an index person has VHL, we wanted
to find out if his family members were expressing
the VHL gene and also to see if subjects with
same genetic makeup in the family could
develop VHL.

COMMUNITY / SOCIAL OPHTHALMOLOGY SESSION
181
If community screening of the genotypes of
affected persons family were carried out and if
they were genotypically normal, no more
screening was necessary for that patient. If
gentypically abnormal, then the screening can be
directed at organ specific involvement like retinal
exam, USG abdomen, CT scan brain, urine VMA
etc to pick up tumors early enough to better the
life expectancy.
Methodology: The study was conducted
during the period May, 2004 to present. We have
identified four families diagnosed clinically with
VHL. Through organized site visits and
outpatient clinic visits, these families was studied
clinically and DNA analysis was carried out
using peripheral blood of known VHL cases and
first-degree relatives. Clinical screening included
CNS imaging where indicated in symptomatic or
known cases, screening abdominal
ultrasonography in all diagnosed cases and firstdegree
relatives, retinal examination by retinal
specialist and measurement of hematocrit.
Molecular biology studies began with the
isolation of genomic DNA from peripheral blood
samples by standard methods. Polymerase chain
reaction (PCR) amplification of microsatellite
markers followed by loss-of-heterozygosity
(LOH) analysis on the denatured PCR products
was done as an initial molecular screening. In the
next phase, PCR amplification of Exons 1, 2, and
3 was carried out followed by (1) purification and
gene sequencing and (2) denaturation and single
strand conformation polymorphism (SSCP)
analysis.
Results
Four families were identified and studied. This
included 14 cases fitting the clinical diagnosis of
VHL. A total of 36 individuals were sampled. In
three of the four families, the inheritance pattern
clearly fit the mendelian autosomal dominant
pattern.
Clinical pattern of VHL: In the four families
studied 69% had cerebellar or brainstem
hemangioblastomas, of which 80% had
undergone multiple surgeries.31 % had renal cell
carcinoma, of which two cases were detected on
routine screening ultrasonography during site
visits. All known cases of VHL had findings of a
multicystic pancreas on the abdominal
ultrasonography. Specifically, there were no
cases of retinal angiomatosis nor polycythemia in
these families. Urine catecholamine analysis was
possible in only one family due to practical
reasons, which revealed no cases of
pheochromocytoma diagnosed.
Genetic analysis: Isolation and PCR
amplification of all three exons of the VHL gene
and subsequent sequence analysis was
successfully carried out in 26 individuals
including 10 of the indexed cases. The base pair
sequence of the VHL gene exons 1, 2, and 3
observed in all indexed cases was identical to the
published gene sequence. No point or frameshift
mutations were detected. Neither SSCP nor LOH
analysis could show any difference in banding
pattern between known cases and controls.
Discussion
In an earlier study in India, 12 angiomas were
decribed in a study, but none of these were
associated with VHL as all the systemic
investigations were negative.They are more
likely to be independent retinal angiomas rather
than VHL. However genetic analysis of these
patients or their family member screening were
not done, depriving us of the knowledge whether
they were a genotypically separate group of
VHL.
In the 4 families and 14 confirmed patients of
VHL which were identified, and genetic and
phenotypic studies done, it was surprising to
note that there were no retinal angiomatosis in
our population. The contrasting western
population showed 41% of cases of VHL having
retinal angiomatosis. A large review of 890 VHL
patients from US showed retinal involvement in
38% and UK registry shows retinal tumors in
41%. The complete absence of retinal tumors in
all 4 families and 14 subjects could be because the
Indian populations studied in Kerala may have
complete deletion of the 3 p gene causing VHL, a
feature which is already decrribed, but much less
common in other populations.
Three genotypic categories were identified for
the retinal tumor, namely amino acid
substitutions, protein truncating mutations and
complete deletions. Complete deletions were
seen in only 7% of the retinal tumors and that is
what we have in the populations studied.
The clinical profile of 14 cases fitting the
diagnosis of VHL syndrome from 4 families is

182 AIOC 2010 PROCEEDINGS
described herein. Specifically, the phenotype
differed from the pattern described in other
countries in that no cases were detected to have
retinal angiomatosis (0%). Predominantly the
combination consisted of CNS hemangioblastomas
(69%), renal cell carcinoma (31%) and
multicystic pancreas – the latter being present in
100 % of cases. Hence, the finding of a multicystic
pancreas in a first degree relative of a known
VHL case, is useful in suspecting the diagnosis
and may eventually prove to be diagnostic. We
hope to cofirm this in the future once the genetic
or chromosomal anomalies are confirmed.
The finding of the normal gene sequence of the
VHL gene in all cases, most likely implicates the
presence of large chromosomal deletions
spanning the VHL locus on chromosome 3p – a
finding which has been described in other
populations. At present fluorescence in situ
hybridization (FISH) analysis is ongoing to
cytogenetically confirm this hypothesis. If
cytogenetic deletions prove to be the cause in all
of our indexed cases, it will have important
implications on the phenotypic variations seen in
our population in comparison to others.
This study was made possible by a grant from the
Kerala State Council for Science, Technology and
Environment.
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Discussion Comment by Dr. Subhash Gupta
The paper was very informative and discussed about VHL disease, a autosomal dominant disease of 14 members
of 4 families. The genetic phenomena and science and symptoms were discussed.Usually, the prognosis is very
poor and ends in total blindness, life expectancy is also poor. He has not discussed about treatment modalities but
thermal photocoagulation, beta ray brachy therapy and anti-vegf theraphy is very helpful nowadays.