RESEARCH ARTICLE Open Access
Th ee no el ge m-line VHL mu a ions in
Hunga ian on Hippel-Lindau pa ien s, including
a nonsense mu a ion in a i een-yea -old boy
wi h enal cell ca cinoma
Ge gely Losonczy
1*
, Fe enc Fazakas
2
, Gyö gy P liegle
3
, Is án Komá omi
2
, E zsébe Balázs
1
, K isz ina Pénzes
2
and And ás Be a
1
Abs ac
Backg ound: Von Hippel-Lindau disease is an au osomal dominan ly inhe i ed highly pene an umo synd ome
p edisposing o e inal and cen al ne ous sys em hemangioblas omas, enal cell ca cinoma and
phaeoch omocy oma among o he less equen complica ions.
Me hods: Molecula gene ic es ing o he VHL gene was pe o med in i e un ela ed amilies a e ced wi h ype I
VHL disease, including se en pa ien s and hei a ailable amily membe s.
Resul s: Molecula gene ic in es iga ions de ec ed h ee no el (c.163 G > T, c.232A > T and c.555C > A causing p.
Glu55X, p.Asn78Ty and p.Ty 185X p o ein changes, espec i ely) and wo p e iously desc ibed (c.340 + 1 G > A and
c.583C > T, esul ing in p.Gly114Asp sX6 and p.195GlnX p o ein changes, espec i ely) ge mline poin mu a ions in
he VHL gene. Molecula modeling o he VHL-ElonginC-HIF-1alpha complex p edic ed ha he p.Asn78Ty amino
acid exchange ema kably al e s he 77-83 loop s uc u e o VHL p o ein and des abilizes he VHL-HIF-1alpha
complex sugges ing ha he mu a ion causes ype I pheno ype and has high isk o associa e o enal cell
ca cinoma. The no el p.55X nonsense mu a ion associa ed o bila e al RCC and e inal angioma in a 15-yea -old
male pa ien .
Conclusion: We desc ibe he ea lies onse enal cell ca cinoma in VHL disease epo ed so a in a 15-yea -old boy
wi h a nonsense VHL mu a ion. Indi idual ailo ing o sc eening schedule based on molecula gene ic s a us should
be conside ed in o de o diagnose se ious complica ions as ea ly as possible. Ou obse a ions add o he
unde s anding o geno ype-pheno ype co ela ion in VHL disease and can be use ul o gene ic counseling and
ollow-up o VHL pa ien s.
Keywo ds: Geno ype-pheno ype co ela ion, Ge mline mu a ion, Renal cell ca cinoma, Von Hippel-Lindau disease
Backg ound
Von Hippel-Lindau (VHL) disease is an au osomal dom-
inan ly inhe i ed highly pene an umo synd ome
a ec ing 1 in 36,000 indi iduals wo ldwide. VHL disease
p edisposes o e inal and cen al ne ous sys em heman-
gioblas omas (HB), enal cell ca cinoma (RCC), phaeoch o-
mocy oma, panc ea ic endoc ine umo s, endolympha ic
sac umo s among o he less equen complica ions. Type
1 diseases is accompanied wi h low isk o phaeoch omocy-
oma, ype IIa is associa ed wi h high isk o phaeoch omo-
cy oma and low isk o enal cell ca cinoma, while ype IIb
is linked o high isk o bo h phaeoch omocy oma and
enal cell ca cinoma. In ype IIc VHL disease only phaeo-
ch omocy oma de elops. Pa ien s ca y a he e ozygous
ge mline mu a ion in he VHL gene. Tumo de elopmen
is ini ia ed by he soma ic inac i a ion o loss o he
emaining wild ype VHL allele [1,2]. VHL p o ein associ-
a es wi h he elongins B and C, cullin2 and Rbx and
* Co espondence: [email p o ec ed]
1
Depa men o Oph halmology, Uni e si y o Deb ecen, Medical and Heal h
Science Cen e , 98. Nagye dei bld, 4012, Deb ecen, Hunga y
Full lis o au ho in o ma ion is a ailable a he end o he a icle
© 2013 Losonczy e al.; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Losonczy e al. BMC Medical Gene ics 2013, 14:3
h p://www.biomedcen al.com/1471-2350/14/3
unc ions as he subs a e ecogni ion componen o an E3-
ubiqui in ligase ha ubiqui ina es HIF-1aplha unde no -
moxic condi ions esul ing in HIF-1alpha p o eolysis [3].
The p o ein model o pVHL19 con ains wo unc ional
subdomains; he be a domain ( esidues 63–154 and esi-
dues 193–204) and he helical alpha-domain ( esidues
155–192). Two impo an binding si es wi hin VHL p o-
ein ha e been iden i ied; one esponsible o elongin C
binding in he alpha domain (amino acid esidues 157–
170) and he o he in he be a domain esponsible o
he binding o HIF1alpha (amino acid esidues 91–113)
[4]. The majo i y o disease-causing missense VHL gene
mu a ions a e loca ed in one o hese wo binding si es.
Molecula gene ic de ec s iden i ied in he backg ound
o he disease ha e g ea ly helped o unde s and he ole
o VHL p o ein in he hypoxia sensing pa hway and o
ecognize geno ype-pheno ype co ela ion, a p e equisi e
o e icien gene ic counseling and pa ien ollow-up [5].
Mo e han 800 di e en ge mline mu a ions a e lis ed in
he VHL mu a ion da abase (h p://www.umd.be:2020/)
[6] and also in a comp ehensi e analysis based on 945
VHL kind eds [7]. He e, we desc ibe 7 membe s o 5
amilies wi h h ee no el and one p e iously epo ed
poin mu a ions in he VHL gene and co ela e gene ic
indings wi h clinical pheno ype.
Me hods
Pa ien s
Se en membe s o i e un ela ed Hunga ian amilies
wi h on Hippel-Lindau disease we e in es iga ed a he
Di ision o Ra e Diseases and he Depa men o Oph-
halmology, a he Uni e si y o Deb ecen in Deb ecen,
Hunga y be ween 1998 and 2011. The diagnosis o VHL
disease was based on physical and oph halmological
examina ions, abdominal CT, c aniospinal and abdominal
MRI and labo a o y es s ( ou ine blood es s and u ine
ca echolamines and anillylmandelic acid). All examina-
ions we e in line wi h p e iously epo ed sc eening p o-
ocols [2,8-10]. One hund ed appa en ly heal hy con ols
we e en olled in he s udy. Con ols, Pa ien s and amily
membe s we e en olled a e ha ing signed in o med con-
sen . All p ocedu es s ic ly adhe ed o he Decla a ion o
Helsinki. App o al was ob ained om local Ins i u ional
E hics Commi ees a he Uni e si y o Deb ecen.
Mu a ion analysis o he VHL gene
Blood samples om pa ien s and amily membe s we e
ob ained a e in o med consen . Genomic DNA was
pu i ied om pe iphe al whi e blood cells. Exons o he
VHL gene we e ampli ied using he ollowing p ime
pai s: 1AF 5
0
-TATAGTGGAAATACAGTAACGAG-3
0
,
1AR 5
0
-GAAGTTGAGCCATACGG-3
0
,1BF5
0
-AGAG
TACGGCCCTGAAGAA-3
0
,1BR5
0
-GCTTACGAGCA
GCGTCAC-3
0
,2F5
0
-ATCTCCTGACCTCATGATCC
-3
0
,2R5
0
- GGGCTTAATTTTTCAAGTGG-3
0
,3F5
0
-
TGAGATCCATCAGTAGTACAGG-3
0
,3R5
0
-CTAAG
GAAGGAACCAGTCC-3
0
. A e an ini ial dena u a ion
s ep a 95°C o 10 minu es, 40 PCR cycles we e pe -
o med unde he ollowing condi ions: dena u a ion a
95°C o 1 minu e, annealing a 56°C o exon 1 and 59°C
o exons 2 and 3 o 1 minu e and ex ension a 72°C o
1 min. An addi ional elonga ion s ep o 7 minu es a 72°C
ollowed he inal cycle. PCR p oduc s pu i ied by ul a-
il a ion we e sequenced by ABIP ism 3100 Gene ic
Analyze (Applied Biosys ems, Fos e Ci y, CA) om bo h
o wa d and e e se p ime s. Clinical and gene ic da a o
p e iously epo ed VHL cases we e ob ained om ci ed
publica ions and om he Uni e sal Mu a ion Da abase
[6]. La ge dele ions we e es ed using he Mul iplex
Liga ion-dependen P obe Ampli ica ion (MLPA) VHL ki
(MRC-Holland, Ams e dam, NL).
E olu iona y alignmen , SIFT analysis and molecula
modeling
Mul iple e olu iona y p o ein sequence alignmen was
pe o med using he open access sou ce o Cons ain -
based Mul iple Alignmen Tool (COBALT) in he case
o he p.N78Y mu a ion [11]. To es ima e whe he his
amino acid subs i u ion a ec s p o ein unc ion we also
applied he online a ailable SIFT analysis ool (h p://si .
jc i.o g/). SIFT p edic ion is based on he deg ee o
conse a ion o amino acid esidues in sequence align-
men s de i ed om closely ela ed sequences, collec ed
h ough PSI-BLAST [12]. We we e cu ious whe he he
SIFT ool could ind any di e ence in he p edic ed e -
ec be ween ype I and ype II missense mu a ions,
he e o e we analyzed all ge mline missense mu a ions
o amilial VHL cases epo ed in he comp ehensi e
analysis o No ds om-O’B ien e al. Six y-six mu a ions
in bo h ype I and II g oups we e analyzed.
Fo molecula modeling, s a ing geome ies o he
model complexes o VHL-, Elongin C- and HIF-1αp o-
eins [13] we e ob ained om he RCSB p o ein da a bank
(pdb ID: 1LM8). The un esol ed N- and C- e minal ag-
men s we e subs i u ed by ace yl and N-me hyl g oups on
he a ailable p o ein agmen s hen he missing hyd ogen
a oms we e added. Molecula dynamics simula ions using
pe iodic bounda y condi ion, explici TIP3P wa e [14]
molecules, AMBER99SB [15,16] o ce ield and addi ional
Na + and Cl- ions (~0.15 M ionic s eng h) we e ca ied
ou wi h bo h he wild- and he Asn78Ty poin mu a ed
VHL p o ein. 4 s ime s ep and 50 ns o al ime ame
was applied o he cons an p essu e (10
5
Pa), cons an
empe a u e (310 K), and cons an pa icle numbe
(68612, 68655 and 68548 o he wild and p.Asn78Ty
mu an sys ems, espec i ely) simula ions. The long ange
elec os a ics o ces we e calcula ed by pa icle mesh
Ewald me hod [17]. The simula ions and he analyses o
Losonczy e al. BMC Medical Gene ics 2013, 14:3 Page 2 o 8
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he ajec o ies we e ca ied ou by means o he G omacs
[18] sui es o so wa e.
Resul s
Molecula gene ic analysis o 11 membe s o 5 un ela ed
VHL amilies de ec ed 3 no el and 2 p e iously desc ibed
poin mu a ions in he VHL gene. No al e a ions could be
de ec ed in he VHL gene wi h he MLPA es . All
mu a ions associa ed o ype I VHL disease. None o he
mu a ions could be obse ed in one hund ed con ol
subjec s. Pheno ype cha ac e is ics and gene ic da a o
he s udy popula ion a e summa ized in Table 1. Elec-
ophe og ams o co esponding VHL mu a ions a e
demons a ed in Figu e 1.
Family A consis ed o wo heal hy pa en s and hei
15-yea -old son, who de eloped bila e al e inal angioma
and se ous e inal de achmen . In sea ch o VHL mani-
es a ions abdominal ul asound scan de ec ed a mass o
18 mm in diame e in he le kidney. Abdominal MRI
scans showed a kidney mass o 15 mm’s in he le kid-
ney and a 10 mm’s diame e mass in he igh kidney. A e
umo enuclea ion, his ological analysis e i ied RCC. MRI
o he c aniospinal axis could no de ec any al e a ions.
No de ia ion om no mal alues was obse ed in he la-
bo a o y es . The pa ien was he e ozygous o he no el
c.163 G > T (p.55GluX) ambe nonsense mu a ion. None
o his pa en s displayed any mu a ions in he VHL gene in-
dica ing a de no o mu a ion.
Two a ec ed p obands o amily B, sis e (index pa-
ien , IP) and b o he , wi h a posi i e amily his o y o
VHL disease we e examined. The sis e de eloped a e -
inal angioma o he igh eye a he age o 48 yea s. MRI
examina ion e ealed a ce ebella hemangioma in bo h
pa ien s. RCC could no be e ealed so a in any o he
pa ien s. Molecula gene ic analysis de ec ed he no el
c.232A > T (p.Asn78Ty ) missense mu a ion in he e ozy-
gous o m in bo h pa ien s.
Family C consis ed o wo pa en s and hei wo daugh-
e s. The 12-yea -old daugh e (IP) was in es iga ed be-
cause o a e inal angioma. A spinal co d hemangioma
was de ec ed on he c aniospinal MRI. No o he mani es-
a ions o VHL disease ha e been de ec ed so a in he
case. Radiological in es iga ions de ec ed bila e al kidney
umo s, a ce ebella and a spinal co d hemangioma in he
34-yea -old a he . His ological analysis o he kidney e i-
ied RCC. Fou yea s la e , e inal angioma de eloped in
bo h o his eyes. The mo he and he o he daugh e we e
ee om any mani es a ions o he disease. Bo h a ec ed
amily membe s we e he e ozygous o he c.340 + 1 G >
A mu a ion, loca ed a he i s exon-in on junc ion. The
mu a ion des oys he conse ed in onic dono splice si e
and mos likely leads o p ema u e p o ein e mina ion (p.
Gly114Asp sX6). The mo he and he heal hy daugh e
showed no mu a ions in he VHL gene. As in ou case, he
mu a ion associa ed o ype I VHL pheno ype in a p e i-
ous epo [19].
The 25-yea -old male pa ien (Pa ien D) was he only
a ailable membe o he amily. His a he had died se -
e al yea s ago due o a cen al ne ous sys em umo ,
p obably a complica ion o VHL disease. The pa ien was
diagnosed wi h ce ebella and spinal co d hemangioma
and a subsequen ly occu ing RCC, while o he mani es a-
ions o VHL disease could no be de ec ed. The no el
he e ozygous nonsense ambe mu a ion (c.555C > A) was
de ec ed in Pa ien D. The mu a ion caused a p e iously
epo ed p o ein unca ion (p.185Ty X) [6,20,21].
Pa ien E su e ed om e inal angioma and ce ebella
hemangioblas oma. No o he VHL- ela ed mani es a ions
could be e ealed so a . Gene ic analysis de ec ed he p e-
iously desc ibed c.583C > T mu a ion esul ing in p ema-
u e p o ein e mina ion (p.195GlnX). In e es ingly, he
mu a ion has been epo ed o associa e o ype I and ype
II VHL disease as well [20,22,23].
SIFT analysis and e olu iona y sequence alignmen
The majo i y o missense mu a ions epo ed in he li -
e a u e in amilial VHL we e p edic ed o be damaging
by SIFT analysis. Howe e , 18% and 35% o he mu a-
ions we e p edic ed o be ole a ed among mu a ions
associa ing o amilial cases o ype I and ype II VHL
Table 1 Summa y o clinical and gene ic indings
Family Pa ien s RCC CNS
HB
Phaeo RA VHL disease
ype
ge mline
mu a ion
localisa ion no el
mu a ion
P edic ed p o ein
modi ica ion
SIFT
analysis
A IP 15 - - 15 1 c.163 G > T exon 1 yes p.Glu55X NA
B IP - 48 - 48 1 c.232A > T exon 1 yes p.Asn78Ty damaging
B o he - 45 - 45 1 c.232A > T exon 1 yes p.Asn78Ty damaging
C IP 14 - 12 1 c.340 + 1 G > A in on 1-2 no p.Gly114Asp sX6 NA
Fa he 34, bila e al 34 - 38 1 c.340 + 1 G > A in on 1-2 no p.Gly114Asp sX6 NA
D IP 25 25 - - 1 c.555C > A exon 3 yes p.Ty 185X NA
E IP - 41 - 41 1 c.583C > T exon 3 no p.195GlnX NA
Numbe s indica e pa ien s’age a de ec ion o he co esponding VHL umo . IP Index pa ien , RCC Clea cell enal cell ca cinoma, CNS HB Cen al ne ous sys em
haemangioblas oma, Phaeo: phaeoch omocy oma, RA Re inal angioma, NA Non-applicable.
Losonczy e al. BMC Medical Gene ics 2013, 14:3 Page 3 o 8
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disease, espec i ely (Fishe ’s exac es , p = 0.048). I is
o be no ed, ha he lowes a e (11%) o ole able mu a-
ions was obse ed in ype I VHL disease associa ing o
RCC. As shown by Figu e 2, he Asn78 amino acid
ep esen s a highly conse ed amino acid among di e -
en species including Homo Sapiens, Pongo pygmeaus,
Ra us no egicus, Mus musculus, Canis amila is and Xen-
opus opicalis. SIFT analysis o he cu en and p e iously
epo ed amino acid exchanges a his posi ion also indi-
ca ed damaging e ec on p o ein unc ion wi h SIFT sco es
o 0, 0, 0, 0.03 and 0.04 in case o con e sion o Ty osine,
His idine, Isoleucin, Se ine and Th eonin, espec i ely.
Figu e 1 Elec ophe og ams ep esen ing VHL mu a ions in he s udy popula ion. Mu a ions a e indica ed by a ows. Panel A: c.163 G > T,
p.Glu55X; Panel B: c.232A > T, p.Asn78Ty ; Panel C: c.340 + 1 G > A, p.Gly114Asp sX6; Panel D: c.555C > A, p.Ty 185X; Panel E: c.583C > T, p.195GlnX.
Losonczy e al. BMC Medical Gene ics 2013, 14:3 Page 4 o 8
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Median conse a ion alues we e below 3.25 in each SIFT
analysis indica ing high con idence p edic ions.
E ec o he Asn78Ty p o ein exchange on he molecula
s uc u e
Molecula simula ion on he model wild ype VHL-
ElonginC - HIF-1alpha complex shows he p o ein com-
plex o be ema kably s able as i is demons a ed by he
i s (Figu e 3A) and las (Figu e 3B) snapsho s om he
co esponding ajec o y. Whe eas compa ing he simu-
la ion ajec o ies o he wild ype and he Asn78Ty
mu an (Figu e 3C) VHL p o eins i is clea ly demon-
s a ed ha his mu a ion ema kably de o ms he 77-83
loop s uc u e in he VHL p o ein. I can be in e p e ed
by he b eaking o he loop-s abilizing H-bond in e -
ac ion be ween Asn78 and and A g82 as well as by a la -
ge space illing p ope y o he y osine side chain. This
de o ma ion sp eads o e he Th 100-A g107 loop o
VHL (ma ked by ed a ow) which inally esul ed in a
weakened in e ac ion be ween his loop and he HIF-
1alpha p o ein (shown by g een a ow). In e es ingly, he
de o med loop s uc u e in VHL de o ms subs an ially
he neighbo ing loop (A g82-Phe93) s uc u e in Elongin
C (shown in blue ellipse) as well. Howe e , his de o m-
a ion did no dis up he in e ac ion be ween VHL and
Elongin C in ou simula ion.
Discussion
We ha e in es iga ed se en VHL pa ien s om i e un-
ela ed Hunga ian amilies. A e de ailed physical, oph-
halmological, adiological and labo a o y in es iga ions
all pa ien s we e ound o ha e ype 1 VHL disease. Mo-
lecula gene ic in es iga ions de ec ed h ee no el and
one p e iously desc ibed poin mu a ions in he VHL
gene. Acco ding o he p esen and a p e ious epo [24],
he e a e 12 VHL amilies in Hunga y iden i ied so a .
Nonsense, missense, ame shi mu a ions and exon dele-
ions we e de ec ed in ou , ou , wo and wo amilies, e-
spec i ely. These p opo ions show no majo de ia ion
om hose o o he popula ions and a e compa able o
p opo ions epo ed by he comp ehensi e analysis o
No ds om-O’B ien e al [7]. The no el c.232A > T (p.
Asn78Ty ) missense mu a ion associa ed o ype I VHL
pheno ype. The si e is e olu iona y conse ed sugges ing
i is an impo an amino acid posi ion o main ain p o ein
s uc u e and unc ion. O he mu a ions a he same
Figu e 2 Mul iple sequence alignmen o VHL p o ein. The Asn78 amino acid is shown in ed. Aspa agine amino acid in his posi ion is
highly conse ed among di e en species.
Figu e 3 Ribbon and balls and s icks ende ing o a ep esen a i e snapsho om molecula simula ions ca ied ou o model VHL-
Elongin C-HIF-1 alpha complexes. The ibbon model o VHL and Elongin C p o ein agmen s a e colo ed o ange and pu ple, espec i ely,
while he agmen o HIF-1αp o ein is shown in g een. The i s and las snapsho s om he simula ion ajec o y o he complex including wild
ype VHL p o ein indica es a ema kably s able p o ein complex (A,B). The Asn78Ty mu a ion (C) ema kably de o ms he 77-83 loop s uc u e in
he VHL p o ein. This de o ma ion sp eads o e he Th 100-A g107 loop o VHL (ma ked by ed a ow) which inally esul s in a weakened
in e ac ion be ween his loop and he HIF-1alpha p o ein (shown by g een a ow). The de o med loop s uc u e in VHL de o ms subs an ially he
neighbo ing loop (A g82-Phe93) s uc u e in Elongin C (shown in blue ellipse in Figu e 3B and Figu e 3C). A om ype colo ing (C, N, O, H a oms
a e colo ed g ey, blue, ed and whi e, espec i ely) was used o he balls and s icks model which we e applied o esidues which a e supposed
o play key ole in in e chain in e ac ions.
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codon, namely p.Asn78His, p.Asn78Se , p.Asn78Th and
p.Asn78Ile associa ed wi h ype I disease pheno ype as
well [20,22,23,25-29]. This mu a ion si e is bu ied wi hin
he p o ein co e whe e mu a ions we e ound o associa e
wi h lowe isk o phaeoch omocy oma compa ed o p o-
ein su ace missense mu a ions [23]. N78H, N78I and
N78S missense mu a ions we e all epo ed o coincide
wi h RCC, howe e he N78T mu a ion did no show his
associa ion [20,22,23,25-29]. Acco ding o a la e publica-
ion RCC associa es wi h high isk o missense mu a ions
loca ed be ween codon 74 and 90 [30]. The e ec o he
no el p.Asn78Ty mu a ion on VHL p o ein s uc u e was
assessed using molecula modeling. The Asn78 side chain
pa icipa es in he s abiliza ion o he u n whe e i can be
ound. The b eaking o he loop-s abilizing H-bond in e -
ac ion be ween Asn78 and A g82 as well as he eplace-
men o his esidue wi h a la ge one ob iously
des abilizes his u n (Figu e 3). I can cause pa ial o glo-
bal mis olding. The mos ema kable e ec o his s uc-
u al eo ganiza ion on p o ein in e ac ions is he high
p obabili y o dissocia ion o he VHL and HIF-1alpha p o-
eins. Despi e he u n de o ma ion, dis up ion o he
VHL-Elongin C in e ac ion could no be obse ed du ing
he molecula dynamics simula ion (Figu e 3). In i o ana-
lysis o pVHL mu an s showed educed abili y o bind and
ubiqui ina e HIF-1alpha in case o ype I, IIa and IIb mu a-
ions, on con a y, mu a ions associa ed o IIc pheno ype
showed no mal binding and ubiqui ina ion o HIF-1alpha
p o ein. Comple ely absen HIF-1alpha binding was only
obse ed in consequence o mu a ions associa ing o RCC
in his s udy [31]. Howe e , a ecen compu a ional s udy
could no con i m he dose-dependen e ec o VHL-HIF-
1alpha dissocia ion and sugges s ha he canonical con ig-
u a ion o he wild- ype be a domain is i al o he
e icien unc ioning o he complex and ha mu a ion o
any o he esidues implica ed in he H-bond ne wo k in
he binding si e dis up s HIF binding [32]. Taken oge he ,
mu a ions in he be a domain dis up ing he VHL-HIF-
1alpha in e ac ion lead o ype I disease pheno ype wi h an
inc eased isk o RCC. Based on hese da a and ou mo-
lecula modeling esul s we can conclude ha he no el p.
Asn78Ty mu a ion associa es o ype I disease and has a
high chance o associa e o RCC as well, while only mino
isk o associa e o phaeoch omocy oma. In an a emp o
assess he e ec o all p e iously epo ed missense mu a-
ions in amilial VHL, we applied SIFT analysis and ound
ha in ype II disease he a e o ole able mu a ions is sig-
ni ican ly highe han in ype I mu a ions. This inding u -
he suppo s ha dele e ious mu a ions dis up ing p o ein
in eg i y a e mo e p one o cause ype I pheno ype. Be-
sides, i sugges s ha SIFT analysis can be a use ul ool
when quick p edic ion o a no el mu a ion is necessa y.
As expec ed, nonsense mu a ions associa ed wi h ype I
VHL pheno ype. Two o he h ee nonsense mu a ions
associa ed o RCC as well. P e ious publica ions show ha
la ge ge mline dele ions, nonsense and ameshi mu a-
ions associa e wi h ype I VHL pheno ype [6,20-22,25,33].
Consis en wi h p e ious publica ions indica ing ha RCC
coincides mo e equen ly wi h nonsense mu a ions han
missense mu a ions, RCC only associa ed o MLTP in ou
pa ien s [23,34]. Nonsense mu a ions independen o hei
exonic loca ion associa e wi h ea lie age a onse and
highe age- ela ed isk o RA and RCC compa ed o mis-
sense mu a ions and la ge dele ions [23]. Suppo ing his
obse a ion, he pa ien wi h he p.55GluX mu a ion was
15-yea s-old when bila e al e inal angioma and bila e al
RCC was diagnosed, which ep esen s he ea lies occu -
ence o RCC in VHL disease epo ed so a [1,35]. VHL
sc eening guidelines [2,8-10] ecommend a sc eening
schedule o VHL pa ien s ega dless o hei gene ic s a-
us in o de o ecognize VHL complica ions in a cu able
s age. The ea lies occu ence o RCC was epo ed in a
six een-yea -old boy. Acco dingly, sc eening guidelines
ecommend yea ly o biannual abdominal MRI om six-
een yea s o age and yea ly abdominal ul asound s a ing
a 8 yea s o age. The occu ence o RCC in a i een-yea s
old boy wi h a unca ing ge mline VHL mu a ion high-
ligh s ha enal cell ca cinoma can be de ec ed in pa ien s
wi h nonsense VHL mu a ions younge han six een yea s
o age. Taken in o accoun ha abdominal MRI scans wi h
a highe esolu ion can de ec RCC ea lie han ul a-
sound, pa ien s wi h nonsense mu a ions known o associ-
a e o RCC and ea ly onse o he VHL disease [23] could
be conside ed o ea lie MRI scans. In gene al, gene ic
s a us migh be conside ed o help ailo ing indi idual
sc eening schedules in he u u e.
Conclusion
Geno ype-pheno ype co ela ion was analyzed and ela ed
o he indings epo ed in p e ious publica ions. In ou
s udy popula ion we con i med ha he dis up ion o he
VHL p o ein in eg i y leading o he dis egula ion o HIF-
1alpha associa e o ype I pheno ype. Acco dingly, we also
showed ha he p opo ion o non-dele e ious mu a ions
is highe in ype II han in ype I missense mu a ions
among mu a ions epo ed in amilial VHL cases. He e we
show he ea lies occu ence o RCC epo ed in VHL dis-
ease so a and sugges indi idual ailo ing o sc eening
schedule based on molecula gene ic s a us. Clinical and
gene ic da a p esen ed in ou s udy can help he ou ine
o gene ic counseling and pa ien ollow-up, as well as he
p esymp oma ic molecula gene ic diagnos ics and con-
ibu e o a be e unde s anding o geno ype-pheno ype
co ela ions.
Abb e ia ions
CNS: Cen al ne ous sys em; MLTP: Mu a ion leading o p o ein unca ion;
Phaeo: Phaeoch omocy oma; RA: Re inal angioma; RCC: Renal cell ca cinoma;
VHL: Von Hippel-Lindau.
Losonczy e al. BMC Medical Gene ics 2013, 14:3 Page 6 o 8
h p://www.biomedcen al.com/1471-2350/14/3
Compe ing in e es s
The au ho s decla e ha hey ha e no compe ing in e es s.
Au ho s’con ibu ions
Acquisi ion o clinical da a: AB, GL, EB, GP. FF and KP pe o med he
molecula gene ic analysis. Th ee dimensional p o ein modeling was ca ied
ou by IK. SIFT and Alignmen analysis: GL. Analysis and in e p e a ion o
da a: AB, IK, FF, GL. D a ing o he manusc ip : GL, IK. Re ision o he
manusc ip o impo an in ellec ual con en : GL, FF, GF, EB, AB. S udy
supe ision: AB. All au ho s ead and app o ed he inal manusc ip .
Acknowledgemen s
The au ho s a e indeb ed o Ms Hen ie a B a u o he excellen echnical
assis ance. The s udy was suppo ed by a g an om he Hunga ian Na ional
Resea ch Fund (OTKA K68616). The au ho s hank László Ka dos o he
s a is ical analysis.
Au ho de ails
1
Depa men o Oph halmology, Uni e si y o Deb ecen, Medical and Heal h
Science Cen e , 98. Nagye dei bld, 4012, Deb ecen, Hunga y.
2
Haemos asis,
Th ombosis and Vascula Biology Resea ch G oup o he Hunga ian Academy
o Sciences, Clinical Resea ch Cen e , Uni e si y o Deb ecen, Deb ecen,
Hunga y.
3
Di ision o Ra e Diseases, Medical and Heal h Science Cen e ,
Uni e si y o Deb ecen, Deb ecen, Hunga y.
Recei ed: 31 July 2012 Accep ed: 28 Decembe 2012
Published: 8 Janua y 2013
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doi:10.1186/1471-2350-14-3
Ci e his a icle as: Losonczy e al.:Th ee no el ge m-line VHL mu a ions
in Hunga ian on Hippel-Lindau pa ien s, including a nonsense
mu a ion in a i een-yea -old boy wi h enal cell ca cinoma. BMC
Medical Gene ics 2013 14:3.
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