Three novel germ-line VHL mutations in Hungarian von Hippel-Lindau patients, including a nonsense mutation in a fifteen-year-old boy with renal cell carcinoma.

1.2 MB·English

by  Losonczy, Gergely

Losonczyetal.BMCMedicalGenetics2013,14:3
http://www.biomedcentral.com/1471-2350/14/3
RESEARCH ARTICLE Open Access
Three novel germ-line VHL mutations in
Hungarian von Hippel-Lindau patients, including
a nonsense mutation in a fifteen-year-old boy
with renal cell carcinoma
Gergely Losonczy1*, Ferenc Fazakas2, György Pfliegler3, István Komáromi2, Erzsébet Balázs1, Krisztina Pénzes2
and András Berta1
Abstract
Background: VonHippel-Lindau disease is an autosomal dominantly inherited highly penetrant tumorsyndrome
predisposing to retinal and central nervous system hemangioblastomas, renal cell carcinoma and
phaeochromocytoma among other less frequent complications.
Methods: Molecular genetic testing of theVHL gene was performed in five unrelated families affetced with type I
VHLdisease,including seven patients and their available family members.
Results: Molecular genetic investigations detected threenovel(c.163 G>T, c.232A>T and c.555C>A causing p.
Glu55X, p.Asn78Tyrand p.Tyr185X protein changes, respectively)and two previously described (c.340+1G>A and
c.583C>T, resulting in p.Gly114AspfsX6 and p.195GlnX protein changes, respectively) germline point mutations in
theVHL gene. Molecular modeling of theVHL-ElonginC-HIF-1alpha complex predicted that thep.Asn78Tyramino
acid exchangeremarkably alters the 77-83 loop structure of VHL protein and destabilizes the VHL-HIF-1alpha
complex suggesting that the mutation causes typeI phenotype and has high risk to associate to renal cell
carcinoma. The novel p.55X nonsense mutation associated to bilateral RCC and retinalangioma ina 15-year-old
malepatient.
Conclusion: We describe theearliestonset renal cell carcinoma in VHLdisease reported so far ina 15-year-old boy
witha nonsenseVHL mutation.Individual tailoring ofscreeningschedulebased onmolecular genetic status should
be considered inorder to diagnose serious complicationsas early as possible. Our observationsadd to the
understanding of genotype-phenotype correlation inVHL disease and can be useful for geneticcounseling and
follow-up ofVHL patients.
Keywords: Genotype-phenotype correlation, Germline mutation, Renal cell carcinoma,VonHippel-Lindau disease
Background sac tumors among other less frequent complications. Type
Von Hippel-Lindau (VHL) disease is an autosomal dom- 1diseasesisaccompaniedwithlowriskofphaeochromocy-
inantly inherited highly penetrant tumor syndrome toma,typeIIaisassociatedwithhighriskofphaeochromo-
affecting 1 in 36,000 individuals worldwide. VHL disease cytomaandlowriskofrenalcellcarcinoma,whiletypeIIb
predisposes to retinal and central nervous system heman- is linked to high risk of both phaeochromocytoma and
gioblastomas(HB),renalcellcarcinoma(RCC),phaeochro- renal cell carcinoma. In type IIc VHL disease only phaeo-
mocytoma, pancreatic endocrine tumors, endolymphatic chromocytoma develops. Patients carry a heterozygous
germline mutation in the VHL gene. Tumor development
is initiated by the somatic inactivation or loss of the
*Correspondence:[email protected]
1DepartmentofOphthalmology,UniversityofDebrecen,MedicalandHealth remaining wild typeVHL allele [1,2]. VHL protein associ-
ScienceCenter,98.Nagyerdeibld,4012,Debrecen,Hungary ates with the elongins B and C, cullin2 and Rbx and
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Losonczyetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Losonczyetal.BMCMedicalGenetics2013,14:3 Page2of8
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0 0 0 0
functionsasthesubstraterecognitioncomponentofanE3- -3, 2R 5- GGGCTTAATTTTTCAAGTGG-3, 3 F 5-
0 0
ubiquitin ligase that ubiquitinates HIF-1aplha under nor- TGAGATCCATCAGTAGTACAGG-3, 3R 5- CTAAG
0
moxicconditionsresultinginHIF-1alphaproteolysis[3]. GAAGGAACCAGTCC-3. After an initial denaturation
The protein model of pVHL19 contains two functional step at 95°C for 10 minutes, 40 PCR cycles were per-
subdomains; the beta domain (residues 63–154 and resi- formed under the following conditions: denaturation at
dues 193–204) and the helical alpha-domain (residues 95°Cfor 1 minute,annealingat 56°Cfor exon 1 and59°C
155–192). Two important binding sites within VHL pro- for exons 2 and 3 for 1 minute and extension at 72°C for
tein have been identified; one responsible for elongin C 1min.Anadditionalelongationstepfor7minutesat72°C
binding in the alpha domain (amino acid residues 157– followed the final cycle. PCR products purified by ultra-
170) and the other in the beta domain responsible for filtration were sequenced by ABIPrism 3100 Genetic
the binding of HIF1alpha (amino acid residues 91–113) Analyzer(AppliedBiosystems,FosterCity,CA)fromboth
[4]. The majority of disease-causing missense VHL gene forward and reverse primers. Clinical and genetic data of
mutations are located in one of these two binding sites. previously reported VHL cases were obtained from cited
Molecular genetic defects identified in the background publications and from the Universal Mutation Database
of the disease have greatly helped to understand the role [6]. Large deletions were tested using the Multiplex
of VHL protein in the hypoxia sensing pathway and to Ligation-dependent Probe Amplification(MLPA)VHL kit
recognize genotype-phenotype correlation, a prerequisite (MRC-Holland,Amsterdam,NL).
of efficient genetic counseling and patient follow-up [5].
More than 800 different germline mutations are listed in Evolutionaryalignment,SIFTanalysisandmolecular
the VHL mutation database (http://www.umd.be:2020/) modeling
[6] and also in a comprehensive analysis based on 945 Multiple evolutionary protein sequence alignment was
VHL kindreds [7]. Here, we describe 7 members of 5 performed using the open access source of Constraint-
families with three novel and one previously reported based Multiple Alignment Tool (COBALT) in the case
point mutations in the VHL gene and correlate genetic of the p.N78Y mutation [11]. To estimate whether this
findingswith clinicalphenotype. amino acid substitution affects protein function we also
applied the onlineavailable SIFTanalysis tool (http://sift.
Methods jcvi.org/). SIFT prediction is based on the degree of
Patients conservation of amino acid residues in sequence align-
Seven members of five unrelated Hungarian families ments derived from closely related sequences, collected
with von Hippel-Lindau disease were investigated at the through PSI-BLAST [12]. We were curious whether the
Division of Rare Diseases and the Department of Oph- SIFT tool could find any difference in the predicted ef-
thalmology, at the University of Debrecen in Debrecen, fect between type I and type II missense mutations,
Hungary between 1998 and 2011. The diagnosis of VHL therefore we analyzed all germline missense mutations
disease was based on physical and ophthalmological of familial VHL cases reported in the comprehensive
examinations, abdominal CT, craniospinal and abdominal analysis of Nordstrom-O’Brien et al. Sixty-six mutations
MRI and laboratory tests (routine blood tests and urine inbothtype IandIIgroups wereanalyzed.
catecholamines and vanillylmandelic acid). All examina- For molecular modeling, starting geometries for the
tions were in line with previously reported screening pro- model complexes of VHL-, Elongin C- and HIF-1α pro-
tocols [2,8-10]. One hundred apparently healthy controls teins[13]wereobtainedfromtheRCSBproteindatabank
were enrolled in the study. Controls, Patients and family (pdb ID: 1LM8). The unresolved N- and C-terminal frag-
memberswereenrolledafterhavingsignedinformedcon- mentsweresubstitutedbyacetylandN-methylgroupson
sent. All procedures strictly adhered to the Declaration of theavailableproteinfragmentsthenthemissinghydrogen
Helsinki. Approval was obtained from local Institutional atoms wereadded. Moleculardynamics simulations using
EthicsCommitteesattheUniversityofDebrecen. periodic boundary condition, explicit TIP3P water [14]
molecules, AMBER99SB [15,16] force field and additional
MutationanalysisoftheVHLgene Na+and Cl- ions (~0.15 M ionic strength) were carried
Blood samples from patients and family members were out with both the wild- and the Asn78Tyr point mutated
obtained after informed consent. Genomic DNA was VHL protein. 4 fs time step and 50 ns total time frame
purified from peripheral white blood cells. Exons of the was applied for the constant pressure (105 Pa), constant
VHL gene were amplified using the following primer temperature (310 K), and constant particle number
0 0
pairs: 1AF 5-TATAGTGGAAATACAGTAACGAG-3, (68612, 68655 and 68548 for the wild and p.Asn78Tyr
0 0 0
1AR 5-GAAGTTGAGCCATACGG-3, 1BF 5- AGAG mutant systems,respectively)simulations. Thelong range
0 0
TACGGCCCTGAAGAA-3, 1BR 5-GCTTACGAGCA electrostatics forces were calculated by particle mesh
0 0
GCGTCAC-3, 2 F 5-ATCTCCTGACCTCATGATCC Ewald method [17]. The simulations and the analyses of
Losonczyetal.BMCMedicalGenetics2013,14:3 Page3of8
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thetrajectorieswerecarriedoutbymeansoftheGromacs was detected on the craniospinal MRI. No other manifes-
[18]suitesofsoftware. tations of VHL disease have been detected so far in her
case. Radiological investigations detected bilateral kidney
tumors,acerebellarandaspinalcordhemangiomainher
Results
34-year-oldfather.Histologicalanalysisofthekidneyveri-
Molecular genetic analysis of 11 members of 5 unrelated fied RCC. Four years later, retinal angioma developed in
VHL familiesdetected 3 noveland 2 previously described bothofhiseyes.Themotherandtheotherdaughterwere
pointmutationsintheVHLgene.Noalterationscouldbe free from any manifestations of the disease. Both affected
detected in the VHL gene with the MLPA test. All family members were heterozygous for the c.340+1 G>
mutationsassociatedtotypeIVHLdisease.Noneofthe A mutation, located at the first exon-intron junction. The
mutations could be observed in one hundred control mutationdestroystheconservedintronicdonorsplicesite
subjects. Phenotype characteristics and genetic data of andmostlikelyleadstoprematureproteintermination(p.
the study population are summarized in Table 1. Elec- Gly114AspfsX6). The mother and the healthy daughter
tropherograms of corresponding VHL mutations are showednomutationsintheVHLgene.Asinourcase,the
demonstratedinFigure1. mutation associated to type I VHL phenotype in a previ-
Family A consisted of two healthy parents and their ousreport[19].
15-year-old son, who developed bilateral retinal angioma The 25-year-old male patient (Patient D) was the only
and serous retinal detachment. In search for VHL mani- available member of the family. His father had died sev-
festations abdominal ultrasound scan detected a mass of eral years ago due to a central nervous system tumor,
18 mm in diameter in the left kidney. Abdominal MRI probably a complication of VHL disease. The patient was
scans showed a kidney mass of 15 mm’s in the left kid- diagnosed with cerebellar and spinal cord hemangioma
neyanda10mm’sdiametermassintherightkidney.After andasubsequentlyoccurringRCC,whileothermanifesta-
tumor enucleation, histological analysis verified RCC. MRI tions of VHL disease could not be detected. The novel
of the craniospinal axis could not detect any alterations. heterozygous nonsense amber mutation (c.555C>A) was
No deviation from normal values was observed in the la- detected in Patient D. The mutation caused a previously
boratory test. The patient was heterozygous for the novel reportedproteintruncation(p.185TyrX)[6,20,21].
c.163 G>T (p.55GluX) amber nonsense mutation. None Patient E suffered from retinal angioma and cerebellar
ofhisparentsdisplayedanymutationsintheVHLgenein- hemangioblastoma. No other VHL-related manifestations
dicatingadenovomutation. couldberevealedsofar.Geneticanalysisdetectedthepre-
Two affected probands of family B, sister (index pa- viouslydescribedc.583C>Tmutationresultinginprema-
tient, IP) and brother, with a positive family history of ture protein termination (p.195GlnX). Interestingly, the
VHL disease were examined. The sister developed a ret- mutationhasbeenreportedtoassociatetotypeIandtype
inal angioma of the right eye at the age of 48 years. MRI IIVHLdiseaseaswell[20,22,23].
examination revealed a cerebellar hemangioma in both
patients. RCC could not be revealed so far in any of the
patients. Molecular genetic analysis detected the novel SIFTanalysisandevolutionarysequencealignment
c.232A>T (p.Asn78Tyr) missense mutation in heterozy- The majority of missense mutations reported in the lit-
gous forminboth patients. erature in familial VHL were predicted to be damaging
FamilyC consistedoftwo parents andtheirtwodaugh- by SIFT analysis. However, 18% and 35% of the muta-
ters. The 12-year-old daughter (IP) was investigated be- tions were predicted to be tolerated among mutations
cause of a retinal angioma. A spinal cord hemangioma associating to familial cases of type I and type II VHL
Table1Summaryofclinicalandgeneticfindings
Family Patients RCC CNS Phaeo RA VHLdisease germline localisation novel Predictedprotein SIFT
HB type mutation mutation modification analysis
A IP 15 - - 15 1 c.163G>T exon1 yes p.Glu55X NA
B IP - 48 - 48 1 c.232A>T exon1 yes p.Asn78Tyr damaging
Brother - 45 - 45 1 c.232A>T exon1 yes p.Asn78Tyr damaging
C IP 14 - 12 1 c.340+1G>A intron1-2 no p.Gly114AspfsX6 NA
Father 34,bilateral 34 - 38 1 c.340+1G>A intron1-2 no p.Gly114AspfsX6 NA
D IP 25 25 - - 1 c.555C>A exon3 yes p.Tyr185X NA
E IP - 41 - 41 1 c.583C>T exon3 no p.195GlnX NA
Numbersindicatepatients’ageatdetectionofthecorrespondingVHLtumor.IPIndexpatient,RCCClearcellrenalcellcarcinoma,CNSHBCentralnervoussystem
haemangioblastoma,Phaeo:phaeochromocytoma,RARetinalangioma,NANon-applicable.
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Figure1ElectropherogramsrepresentingVHLmutationsinthestudypopulation.Mutationsareindicatedbyarrows.PanelA:c.163G>T,
p.Glu55X;PanelB:c.232A>T,p.Asn78Tyr;PanelC:c.340+1G>A,p.Gly114AspfsX6;PanelD:c.555C>A,p.Tyr185X;PanelE:c.583C>T,p.195GlnX.
disease, respectively (Fisher’s exact test, p=0.048). It is Rattusnorvegicus,Musmusculus,CanisfamilarisandXen-
tobenoted,thatthelowestrate (11%)oftolerable muta- opus tropicalis. SIFTanalysis of the current and previously
tions was observed in type I VHL disease associating to reported amino acid exchanges at this position also indi-
RCC. As shown by Figure 2, the Asn78 amino acid cateddamagingeffectonproteinfunctionwithSIFTscores
represents a highly conserved amino acid among differ- of 0, 0, 0, 0.03 and 0.04 in case of conversion to Tyrosine,
ent species including Homo Sapiens, Pongo pygmeaus, Histidine, Isoleucin, Serine and Threonin, respectively.
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Figure2MultiplesequencealignmentofVHLprotein.TheAsn78aminoacidisshowninred.Asparagineaminoacidinthispositionis
highlyconservedamongdifferentspecies.
Median conservation values were below 3.25 in each SIFT C (shown in blue ellipse) as well. However, this deform-
analysisindicatinghighconfidencepredictions. ation did not disrupt the interaction between VHL and
Elongin Cinoursimulation.
EffectoftheAsn78Tyrproteinexchangeonthemolecular
structure Discussion
Molecular simulation on the model wild type VHL- We have investigated seven VHL patients from five un-
ElonginC - HIF-1alpha complex shows the protein com- related Hungarian families. After detailed physical, oph-
plex to be remarkably stable as it is demonstrated by the thalmological, radiological and laboratory investigations
first (Figure 3A) and last (Figure 3B) snapshots from the all patients were found to have type 1 VHL disease. Mo-
corresponding trajectory. Whereas comparing the simu- lecular genetic investigations detected three novel and
lation trajectories for the wild type and the Asn78Tyr one previously described point mutations in the VHL
mutant (Figure 3C) VHL proteins it is clearly demon- gene.Accordingtothepresentandapreviousreport[24],
strated that this mutation remarkably deforms the 77-83 there are 12 VHL families in Hungary identified so far.
loop structure in the VHL protein. It can be interpreted Nonsense,missense,frameshiftmutationsandexondele-
by the breaking of the loop-stabilizing H-bond inter- tionsweredetectedinfour,four,twoandtwofamilies,re-
action between Asn78 andand Arg82 as well as bya lar- spectively. These proportions show no major deviation
ger space filling property of the tyrosine side chain. This from those of other populations and are comparable to
deformation spreads over the Thr100-Arg107 loop of proportions reported by the comprehensive analysis of
VHL (marked by red arrow) which finally resulted in a Nordstrom-O’Brien et al [7]. The novel c.232A>T (p.
weakened interaction between this loop and the HIF- Asn78Tyr) missense mutation associated to type I VHL
1alpha protein(shownbygreen arrow). Interestingly, the phenotype. The site is evolutionary conserved suggesting
deformed loop structure in VHL deforms substantially itisanimportantaminoacidpositiontomaintainprotein
theneighboringloop (Arg82-Phe93)structureinElongin structure and function. Other mutations at the same
Figure3RibbonandballsandsticksrenderingofarepresentativesnapshotfrommolecularsimulationscarriedoutformodelVHL-
ElonginC-HIF-1alphacomplexes.TheribbonmodelofVHLandElonginCproteinfragmentsarecoloredorangeandpurple,respectively,
whilethefragmentofHIF-1αproteinisshowningreen.Thefirstandlastsnapshotsfromthesimulationtrajectoryofthecomplexincludingwild
typeVHLproteinindicatesaremarkablystableproteincomplex(A,B).TheAsn78Tyrmutation(C)remarkablydeformsthe77-83loopstructurein
theVHLprotein.ThisdeformationspreadsovertheThr100-Arg107loopofVHL(markedbyredarrow)whichfinallyresultsinaweakened
interactionbetweenthisloopandtheHIF-1alphaprotein(shownbygreenarrow).ThedeformedloopstructureinVHLdeformssubstantiallythe
neighboringloop(Arg82-Phe93)structureinElonginC(showninblueellipseinFigure3BandFigure3C).Atomtypecoloring(C,N,O,Hatoms
arecoloredgrey,blue,redandwhite,respectively)wasusedfortheballsandsticksmodelwhichwereappliedforresidueswhicharesupposed
toplaykeyroleininterchaininteractions.
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codon, namely p.Asn78His, p.Asn78Ser, p.Asn78Thr and associatedtoRCCaswell.Previouspublicationsshowthat
p.Asn78Ile associated with type I disease phenotype as large germline deletions, nonsense and frameshift muta-
well [20,22,23,25-29]. This mutation site is buried within tionsassociatewithtypeIVHLphenotype[6,20-22,25,33].
theproteincorewheremutationswerefound toassociate ConsistentwithpreviouspublicationsindicatingthatRCC
withlower risk of phaeochromocytomacompared topro- coincides more frequently with nonsense mutations than
tein surface missense mutations [23]. N78H, N78I and missensemutations,RCConlyassociatedtoMLTPinour
N78S missense mutations were all reported to coincide patients[23,34].Nonsensemutationsindependentoftheir
withRCC,howevertheN78Tmutationdidnot showthis exonic location associate with earlier age at onset and
association [20,22,23,25-29]. According to a later publica- higher age-related risk of RA and RCC compared to mis-
tion RCC associates with high risk to missense mutations sense mutations and large deletions [23]. Supporting this
located between codon 74 and 90 [30]. The effect of the observation, the patient with the p.55GluX mutation was
novelp.Asn78TyrmutationonVHLproteinstructurewas 15-years-old when bilateral retinal angioma and bilateral
assessedusingmolecularmodeling.TheAsn78side chain RCC was diagnosed, which represents the earliest occur-
participatesinthestabilizationoftheturnwhereitcanbe rence of RCC in VHL disease reported so far [1,35]. VHL
found. The breaking of the loop-stabilizing H-bond inter- screening guidelines [2,8-10] recommend a screening
action between Asn78 and Arg82 as well as the replace- schedule for VHL patients regardless of their genetic sta-
ment of this residue with a larger one obviously tus in order to recognizeVHL complications in a curable
destabilizesthisturn(Figure3).Itcancausepartialorglo- stage. The earliest occurrence of RCC was reported in a
bal misfolding. The most remarkable effect of this struc- sixteen-year-old boy. Accordingly, screening guidelines
tural reorganization on protein interactions is the high recommend yearly or biannual abdominal MRI from six-
probabilityofdissociationoftheVHLandHIF-1alphapro- teenyearsofageandyearlyabdominalultrasoundstarting
teins. Despite the turn deformation, disruption of the at8yearsofage.TheoccurrenceofRCCinafifteen-years
VHL-Elongin C interaction could not be observed during old boy with a truncating germline VHL mutation high-
themoleculardynamicssimulation(Figure3).Invitroana- lightsthatrenalcellcarcinomacanbedetectedinpatients
lysisofpVHLmutantsshowedreducedabilitytobindand withnonsenseVHL mutationsyounger thansixteen years
ubiquitinateHIF-1alphaincaseoftypeI,IIaandIIbmuta- ofage.TakenintoaccountthatabdominalMRIscanswith
tions, on contrary, mutations associated to IIc phenotype a higher resolution can detect RCC earlier than ultra-
showed normal binding and ubiquitination of HIF-1alpha sound,patientswithnonsensemutationsknowntoassoci-
protein. Completely absent HIF-1alpha binding was only ate to RCC and early onset oftheVHL disease [23] could
observed in consequence of mutations associating to RCC be considered for earlier MRI scans. In general, genetic
in this study [31]. However, a recent computational study status might be considered to help tailoring individual
couldnotconfirmthedose-dependenteffectofVHL-HIF- screeningschedulesinthefuture.
1alphadissociationandsuggeststhatthe canonicalconfig-
uration of the wild-type beta domain is vital for the Conclusion
efficient functioning of the complex and that mutation of Genotype-phenotypecorrelationwasanalyzedandrelated
any of the residues implicated in the H-bond network in to the findings reported in previous publications. In our
the binding sitedisruptsHIF binding [32]. Taken together, study population we confirmed that the disruption of the
mutations in the beta domain disrupting the VHL-HIF- VHLproteinintegrityleadingtothedisregulationofHIF-
1alphainteractionleadtotypeIdiseasephenotypewithan 1alphaassociatetotypeIphenotype.Accordingly,wealso
increased risk of RCC. Based on these data and our mo- showed that the proportion of non-deleterious mutations
lecularmodelingresultswe canconcludethatthenovel p. is higher in type II than in type I missense mutations
Asn78Tyr mutation associates to type I disease and has a amongmutationsreportedinfamilialVHLcases.Herewe
high chancetoassociate to RCC as well, while onlyminor showtheearliestoccurrenceofRCCreportedinVHLdis-
risk to associate to phaeochromocytoma. In an attempt to ease so far and suggest individual tailoring of screening
assess the effect of all previously reported missense muta- schedule based on molecular genetic status. Clinical and
tions in familial VHL, we applied SIFTanalysis and found genetic data presented in our study can help the routine
thatintypeIIdiseasetherateoftolerablemutationsissig- ofgeneticcounselingandpatientfollow-up,aswellasthe
nificantlyhigherthanintypeImutations.Thisfindingfur- presymptomatic molecular genetic diagnostics and con-
thersupportsthatdeleteriousmutationsdisruptingprotein tribute to a better understanding of genotype-phenotype
integrity are more prone to cause type I phenotype. Be- correlations.
sides, it suggests that SIFT analysis can be a useful tool
whenquickpredictionofanovelmutationisnecessary. Abbreviations
CNS:Centralnervoussystem;MLTP:Mutationleadingtoproteintruncation;
As expected, nonsense mutations associated with type I
Phaeo:Phaeochromocytoma;RA:Retinalangioma;RCC:Renalcellcarcinoma;
VHL phenotype. Two of the three nonsense mutations VHL:VonHippel-Lindau.
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Acquisitionofclinicaldata:AB,GL,EB,GP.FFandKPperformedthe
19. OlschwangS,RichardS,BoissonC,GiraudS,PuigPL,RescheF,ThomasG:
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data:AB,IK,FF,GL.Draftingofthemanuscript:GL,IK.Revisionofthe
20. CrosseyPA,RichardsFM,FosterK,GreenJS,ProwseA,LatifF,LermanMI,
manuscriptforimportantintellectualcontent:GL,FF,GF,EB,AB.Study
ZbarB,AffaraNA,Ferguson-SmithMA,etal:Identificationofintragenic
supervision:AB.Allauthorsreadandapprovedthefinalmanuscript.
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TheauthorsareindebtedtoMsHenriettaBratuforherexcellenttechnical 21. StolleC,GlennG,ZbarB,HumphreyJS,ChoykeP,WaltherM,PackS,Hurley
assistance.ThestudywassupportedbyagrantfromtheHungarianNational K,AndreyC,KlausnerR,LinehanWM:Improveddetectionofgermline
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1DepartmentofOphthalmology,UniversityofDebrecen,MedicalandHealth HP,TishermanS,MulvihillJJ,GrossDJ,ShuinT,WhaleyJ,SeizingerB,KleyN,
ScienceCenter,98.Nagyerdeibld,4012,Debrecen,Hungary.2Haemostasis, OlschwangS,BoissonC,RichardS,LipsCH,LermanM,etal:Germline
ThrombosisandVascularBiologyResearchGroupoftheHungarianAcademy mutationsintheVonHippel-Lindaudisease(VHL)geneinfamiliesfrom
ofSciences,ClinicalResearchCenter,UniversityofDebrecen,Debrecen, NorthAmerica,Europe,andJapan.HumMutat1996,8:348–357.
Hungary.3DivisionofRareDiseases,MedicalandHealthScienceCenter, 23. OngKR,WoodwardER,KillickP,LimC,MacdonaldF,MaherER:Genotype-
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Received:31July2012Accepted:28December2012 24. GergicsP,PatocsA,TothM,IgazP,SzucsN,LikoI,FazakasF,SzaboI,
Published:8January2013 KovacsB,GlazE,RaczK:GermlineVHLgenemutationsinHungarian
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