Ser80Ile mutation and a concurrent Pro25Leu variant of the VHL gene in an extended Hungarian von Hippel-Lindau family

BioMed Cen al Page 1 o 8 (page numbe no o ci a ion pu poses) BMC Medical Gene ics Open Access Resea ch a icle Se 80Ile mu a ion and a concu en P o25Leu a ian o he VHL gene in an ex ended Hunga ian on Hippel-Lindau amily A ila Pa ocs1, Pe e Ge gics2, Ka alin Balogh2, Miklos To h2, Fe enc Fazakas3, Is an Liko4 and Ka oly Racz*2 Add ess: 1Molecula Medicine Resea ch G oup, Hunga ian Academy o Sciences and Semmelweis Uni e si y, Szen ki ályi 46, H-1088 Budapes , Hunga y, 22nd Depa men o Medicine, Facul y o Medicine, Semmelweis Uni e si y, Szen ki ályi 46, H-1088 Budapes , Hunga y, 3Medical and Heal h Science Cen e , Uni e si y o Deb ecen, H-4200, Deb ecen, Hunga y and 4Rich e Gedeon LTD, Budapes , Hunga y Email: A ila Pa ocs - [email p o ec ed]; Pe e Ge gics - ge pe @yahoo.com; Ka alin Balogh - bka us@ eemail.hu; Miklos To h - o [email protected] e.hu; Fe enc Fazakas - wol i[email p o ec ed]e.hu; Is an Liko - I.Liko@ ich e .hu; Ka oly Racz* - [email p o ec ed].hu * Co esponding au ho Abs ac Von Hippel-Lindau disease (VHL) is a a e au osomal dominan disease cha ac e ized by de elopmen o cys ic and umo ous lesions a mul iple si es, including he b ain, spinal co d, kidneys, ad enals, panc eas, epididymis and eyes. The clinical pheno ype esul s om molecula abno mali ies o he VHL umo supp esso gene, mapped o human ch omosome 3p25-26. The VHL gene encodes wo unc ionally ac i e VHL p o eins due o he p esence o wo ansla ional ini ia ion si es sepa a ed by 53 codons. The majo i y o disease-causing mu a ions ha e been de ec ed downs eam o he second ansla ional ini ia ion si e, bu he e a e con lic ing da a as o whe he ew mu a ions loca ed in he i s 53 codons, such as he P o25Leu could ha e a pa hogenic ole. In his pape we epo a la ge Hunga ian VHL ype 2 amily consis ing o 32 membe s in whom a disease-causing AGT80AAT (Se 80Ile) c.239G>A, p.Se 80Ile mu a ion, bu no he concu en CCT25CTT (P o25Leu) c.74C>T, p.P o25Leu a ian co-seg ega ed wi h he disease. To ou knowledge, he Se 80Ile mu a ion has no been p e iously desc ibed in VHL ype 2 pa ien s wi h high isk o pheoch omocy oma and enal cell cance . The e o e, his inding ep esen s a no el geno ype-pheno ype associa ion and VHL kind eds wi h Se 80Ile mu a ion will equi e ca e ul su eillance o pheoch omocy oma. We concluded ha he P o25Leu a ian is a a e, neu al a ian , bu he p esence such a a e gene a ian may make gene ic counseling di icul . Backg ound Von Hippel-Lindau disease (VHL) (OMIM n . 193300) is a a e au osomal dominan mul i-o gan disease caused by molecula abno mali ies o he VHL umo supp esso gene [1]. Pa ien s wi h VHL a e a isk o de elopmen o e inal, ce ebella , spinal, panc ea ic and enal heman- gioblas omas, pulmona y and li e hemangiomas, clea - cell enal ca cinomas, pheoch omocy omas, endolym- pha ic sac umo s, mul iple enal, epididymal and panc e- a ic cys s, cys adenomas o he epididymis and o he b oad ligamen , and panc ea ic isle cell umo s [1-3]. Based on he p esence o absence o pheoch omocy oma, wo main sub ypes o he VHL disease ha e been iden i- ied. Pa ien s wi h VHL ype 1 a e a isk o de elop enal Published: 16 Ap il 2008 BMC Medical Gene ics 2008, 9:29 doi:10.1186/1471-2350-9-29 Recei ed: 26 Oc obe 2007 Accep ed: 16 Ap il 2008 This a icle is a ailable om: h p://www.biomedcen al.com/1471-2350/9/29 © 2008 Pa ocs 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. BMC Medical Gene ics 2008, 9:29 h p://www.biomedcen al.com/1471-2350/9/29 Page 2 o 8 (page numbe no o ci a ion pu poses) cell ca cinoma and hemangioblas oma bu p edomi- nan ly wi hou pheoch omocy oma, while hose wi h VHL ype 2 may ha e all mani es a ions o he disease p e- dominan ly including pheoch omocy oma. VHL ype 2 has been subdi ided in o sub ype 2A and 2B, wi h a low and high isk o enal cell ca cinoma, espec i ely, whe eas sub ype 2C is a pheoch omocy oma-only pheno- ype. The p e alence o he VHL disease a ies be ween 1:39,000 in Ge many and 1:53,000 in Eas Anglia [4,5]. Mani es a ions o he disease show a iable exp ession and se e al pa ien s may ha e only one mani es a ion [4]. The human VHL gene maps o ch omosome 3p25-26 [1]. As p edic ed by Knudson's wo-hi model, umo de elop- men equi es inac i a ion o bo h copies; he i s hi , he ge mline mu a ion o dele ion is ollowed by soma ic al e a ions usually de ec ed as loss o he e ozygosi y. The VHL gene has wo ansla ional ini ia ion si es sepa a ed by 53 codons. O he wo p o eins encoded by he VHL gene, he la ge one con ains 213 amino acids (pVHL30), whe eas he sho e p o ein consis s o 160 amino acids (pVHL18). Bo h p o eins a e unc ionally ac i e and hey sha e he same mechanism o umo supp esso ac i i y [6-8]. The VHL p o eins o m a mul ime ic complex wi h Elongin B, Elongin C, Cul2 and Rbx1. This complex is in ol ed in ubiqui in-dependen p o eolysis o la ge cel- lula p o eins ia con olled deg ada ion o α-subuni s o he he e odime ic ansc ip ion ac o hypoxia inducible ac o (HIF) [9,10]. In addi ion, complexes con aining pVHL, Elongin B, Elongin C, Cul2 and Rbx1 a ge p o- eins a e in ol ed in cell-cycle egula ion o deg ada ion, sugges ing ha pVHL has a possible ole in cell-cycle exi [11]. In 1999, S ebbins and co-wo ke s p esen ed he h ee-dimensional p o ein s uc u e o he VHL-ElonginC- ElonginB complex. They iden i ied wo unc ionally ac i e si es; he ElonginC binding si e in he α-helical domain in ol ing esidues be ween amino acids 157 and 170, and he HIF binding si e in he β-shee domain om esidue 91 o 113 [8]. The majo i y o VHL pa ien s ha e dele ions, inse ions, o mu a ions downs eam o he second ansla ional ini ia- ion si e loca ed a codon 54 o he VHL gene. In e es - ingly, missense mu a ions encoding esidues o he p o ein binding si es ha e been associa ed wi h VHL ype 2, whe eas VHL ype 1 is caused ei he by missense o non- sense mu a ions a ec ing he hyd ophobic co e, o by pa - ial gene dele ions which esul in a comple e de ec o p o ein unc ion. I has been also demons a ed ha some mu an pVHL, which a e associa ed wi h he ype 2C phe- no ype, may impai ib onec in ma ix assembly while hey e ain he abili y o down egula e HIF [12]. Because mu a ions a ec ing he i s 53 codons o he VHL gene ha e no e ec on he s uc u e o he sho e VHL p o ein, i seemed pa icula ly in e es ing o cla i y whe he hese mu a ions could exe a pa hogenic e ec . In one s udy he P o25Leu a ian has been associa ed wi h a spo adic pheoch omocy oma [13]. bu o he s ud- ies in ol ing a limi ed numbe o cases ailed o con i m he pa hogenic ole o his a ian [14,15]. In his pape we epo a la ge Hunga ian VHL ype 2 amily wi h a dis- ease-causing AGT80AAT (Se 80Ile) c.239G>A, p.Se 80Ile mu a ion and a concu en CCT25CTT (P o25Leu) c.74C>T, p.P o25Leu a ian (iden i ica ion numbe : s35460768 (dbSNP127) o he VHL gene. We show ha in amily membe s he Se 80Ile mu a ion, bu no he P o25Leu a ian co-seg ega ed wi h he disease. In o de o con i m he pa hogenici y o he Se 80Ile mu a ion and o es whe he he P o25Leu a ian migh be neu al, an e olu iona y mul iple sequence alignmen analysis (MSA) combined wi h a Align GVGD was.pe o med. Th ee- dimensional modeling o he Ile80-mu an p o ein is also p esen ed. Pa ien s and me hods Pa ien s and da a collec ion A la ge Hunga ian VHL ype 2 amily spanning i e gene - a ions and in ol ing 32 membe s (Fig 1) was e alua ed a he 2nd Depa men o Medicine, Facul y o Medicine, Semmelweis Uni e si y in Budapes , Hunga y. Ini ial sc eening included medical his o y, physical examina ion, abdominal ul asonog aphy, abdominal and b ain com- pu ed omog aphy (CT) o magne ic esonance imaging (MRI), oph halmologic examina ion, ou ine biochemi- cal es ing and 24-h u ina y ca echolamine me aboli e de e mina ions. Sc eening o VHL gene mu a ions W i en in o med consen was ob ained om all amily membe s who pa icipa ed in he s udy. Genomic DNA was ex ac ed om pe iphe al blood leukocy es using DNA isola ion ki o mammalian blood samples (Boe- h inge Mannheim Co po a ion, Indianapolis, IN). Mu a- ion analysis o he VHL gene was pe o med as p e iously desc ibed [16]. E olu iona y alignmen analysis o he VHL p o ein Mul iple sequence alignmen (MSA) o p o ein sequences was pe o med using 3D-co ee, a eely a ailable web- based ool o aligning mul iple sequences [17]. A o al o eigh sequences: Mus musculus (ENSMUSG0000003393 3), Ra us no egicus (ENSRNOG00000010258), Canis amilia is (ENSCAFG00000005149), Homo sapiens (EN ST00000256474), Gallus gallus (ENSGALG0000000136 78), and e olu iona y mo e dis an species including D o- sophila melanogas e (CG13221_CG13221-RA), Xeno- pus opicalis (ENSXET00000001448) and Taki ugu ub ipes (SINFRUG00000121189) we e analysed. BMC Medical Gene ics 2008, 9:29 h p://www.biomedcen al.com/1471-2350/9/29 Page 3 o 8 (page numbe no o ci a ion pu poses) One popula me hod o measu ing biochemical dis- ances be ween pai s o amino acids is he G an ham Di - e ence (18), which akes in o accoun he composi ion, pola i y and olume o mu an and wild- ype amino acids. In his s udy, we ha e cons uc ed a VHL p o ein MSA and combined a conse a ion sco e (GV) wi h a measu e o biochemical di e ence be ween wild- ype and mu an esidues wi h espec o he alignmen (GD). This ex ension o he G an ham Di e ence, called Align GVGD, has p e iously been success ully applied o BRCA1 and TP53 genes (19, 20). Align GVGD is eely a ailable online (21). Th ee-dimensional s uc u e modeling o he VHL p o ein The coo dina es o he pVHL we e ob ained om 1 cb_a.pdb s uc u e ile [8]. The h ee-dimensional image was gene a ed wi h Swiss-PdbViewe in combina- ion wi h POV-Ray [22]. Resul s Geno ype-pheno ype associa ions The index pa ien was ope a ed o pheoch omocy oma and enal cell ca cinoma diagnosed a he age o 34 y and 40 y , espec i ely. She also de eloped a b ain heman- gioblas oma a he age o 42 y . Gene ic sc eening indi- ca ed ha she had a he e ozygous AGT80AAT (Se 80Ile) c.239G>A, p.Se 80Ile mu a ion o he VHL gene. The 70- y -old mo he o he index pa ien p o ed o be no only a gene ca ie o he Se 80Ile mu a ion, bu she also had he CCT25CTT (P o25Leu) c.74C>T, p.P o25Leu a ian (iden i ica ion numbe : s35460768 (dbSNP127)), bo h in he e ozygous o ms. Howe e , he mo he showed no clinical, biochemical o adiological e idence o VHL- associa ed umo s. The absence o he P o25Leu a ian in he index pa ien indica ed, ha he Se 80Ile mu a ion and he P o25Leu a ian in he mo he we e p esen in sepa a e alleles. In addi ion o he index pa ien and he mo he , he Se 80Ile mu a ion was de ec ed in o he 5 membe s o he Pedig ee o a Hunga ian kind ed wi h on Hippel-Lindau diseaseFigu e 1 Pedig ee o a Hunga ian kind ed wi h on Hippel-Lindau disease. BMC Medical Gene ics 2008, 9:29 h p://www.biomedcen al.com/1471-2350/9/29 Page 4 o 8 (page numbe no o ci a ion pu poses) amily (III/7, III/8, IV/13, IV/14 and IV/16) (Fig 1), o which all bu one membe (III/7, III/8, IV/13 and IV/14) had clinical mani es a ions o he disease. Impo an ly, pheoch omocy omas we e iden i ied in am- ily membe s III/8 and IV/14, o whom, in pa ien IV/14 his was he i s sign o he disease mani es ed a he age o 10 yea s and in ol ed bo h ad enals. O he ypical mani es a ions included b ain hemangioblas omas in amily membe s: III/7, III/8 and IV/13, e ina hemangiob- las omas in amily membe s III/7, III/8, IV/13 and IV/14, and enal cys s in amily membe IV/13. In addi ion, am- ily his o y e ealed ha one sis e o he mo he o he index pa ien (II/5) died due o bila e al enal cell ca cino- mas. Ano he sis e o he mo he o he index pa ien (II.4) died because o a me as a ic panc ea ic umo wi h- ou his ological con i ma ion and, he e o e, his amily membe was no conside ed as ha ing VHL. The age, clin- ical mani es a ions, and he age a diagnosis o VHL-asso- cia ed lesions in ca ie s o he Se 80Ile mu a ion and in hose wi h clinically p o en VHL-associa ed umo s a e summa ized in Table 1. In addi ion o he mo he o he index pa ien , he P o25Leu a ian was de ec ed on ano he amily membe (III/6) (Fig 1), who was clinically heal hy. The p e alence o he P o25Leu a ian was es ed in 16 membe s o o he 8 VHL amilies and in 39 pa ien s wi h spo adic pheoch omocy omas, bu none o he pa ien s had his a ian . E olu iona y alignmen analysis o he VHL gene As shown in Fig. 2., he Se a amino acid posi ion 80 ep- esen s a highly conse ed amino acid esidue among di - e en species, including a , mouse, dog, chicken and an e olu iona y mo e dis an species, Xenopus opicalis. By con as , he P o a amino acid posi ion 25 ailed o show a conse ed pa e n. Using Align-GVGD c i e ia bo h a - ian s we e p edic ed as neu al ul illing he c i e ia 0<GV ≤ 61,3 and GD>0 ( he calcula ed GV and GD sco es o P o25Leu polymo phism we e 218.82 and 4.86 and o Se 80Ile 219.56 and 3.24, espec i ely). E ec o change o Se 80 o Ile80 on p o ein s uc u e The h ee-dimensional model o he pVHL is a ailable om esidue Me 54, which allowed us o analyse unc- ional consequences o he Se 80Ile mu a ion using a h ee-dimensional compu a ional modeling. The CPK diag am in Fig. 3 illus a es bo h he wild- ype Se 80 and he mu an Ile80 p o eins. The Se 80 esidue makes igh junc ions wi h P o103 and Ile151 by hyd ogen-bonds be ween he amino g oup o Se 80 and he ca boxyl oxy- gen o P o103 and Ile151. The Se 80 esidue is loca ed on he same side whe e ElonginC binds, and esidues P o81 and A g82 a e in ol ed in di ec binding. The Asn78 esi- due om he β-domain, oge he wi h hyd ophobic co e esidues (P o86, Phe76, Phe119, T p117 and Val130) ha e impo an oles o s uc u al in eg i y o he β-sand- wich [8]. A change o Se o Ile a posi ion 80 dis u bs his in eg i y o he β-sandwich due o a change in he o ien a- ion o he A g82 esidue, which dis u bs i s linke ole and, consequen ly, he hyd ogen bond be ween A g82 and Leu153 disappea s. I has been al eady demons a ed ha he A g82 esidue plays a cen al ole in s uc u al in eg i y o he pVHL, as i makes signi ican hyd ogen- bond con ac s wi h Leu153, Val155, Lys159 and A g161 and, h ough hese con ac s; i is in ol ed in in e ac ions be ween pVHL and ElonginC [8]. Discussion In his pape we desc ibe a la ge Hunga ian VHL ype 2 amily consis ing o 32 membe s, in whom a disease-caus- ing AGT80AAT (Se 80Ile) c.239G>A, p.Se 80Ile mu a ion Table 1: Clinical mani es a ions o VHL disease in ca ie s o he Se 80Ile mu a ion and/o he P o25Leu a ian and in hose wi h clinically p o en VHL-associa ed umo s Family membe (age, yea s) Renal cell ca cinoma (age a p esen a ion, yea s) B ain heman- gioblas oma (age a p esen a ion, yea s) Pheoch omo- cy oma (age a p esen a ion, yea s) Re ina heman- gioblas oma (age a p esen a ion, yea s) O he clinical mani es a ions Resul o gene ic sc eening II/1 (70)-----P o25Leu Se 80Ile III/6 (44)-----P o25Leu II/5 (63) + bila e al (45) - - - ND III/1* (42) + bila e al (40) + (42) + (34) - - Se 80Ile III/7 (47) - + (37) - + (18) - Se 80Ile III/8 (39) - + (38) + (39) + (29) - Se 80Ile IV/13 (27) - + (22) - + (23) enal cys s (20) Se 80Ile IV/14 (26) - - + bila e al (10) + (26) - Se 80Ile IV/16 (16)-----Se 80Ile *, index pa ien ; -, p esen ; +, absen ; ND, no de e mined (DNA o gene ic es ing was no a ailable) BMC Medical Gene ics 2008, 9:29 h p://www.biomedcen al.com/1471-2350/9/29 Page 5 o 8 (page numbe no o ci a ion pu poses) and a concu en CCT25CTT (P o25Leu) c.74C>T, p.P o25Leu a ian we e iden i ied. Sc eening o amily membe s indica ed ha 7 amily membe s had he Se 80Ile mu a ion and 2 membe s had he P o25Leu a - ian , and ha he wo gene ic al e a ions we e ansmi ed in sepa a e alleles. Mo e impo an ly, we showed ha he Se 80Ile mu a ion, bu no he P o25Leu a ian co-seg e- ga ed wi h he VHL ype 2 pheno ype and ha he P o25Leu a ian ne e occu ed in amily membe s who had mani es a ions o he disease. Howe e , one amily membe who had bo h gene ic al e a ions and one mem- be wi h he Se 80Ile mu a ion wi hou he P o25Leu a - ian showed no mani es a ions o he disease, sugges ing ha his a ian is no pa hogenic o VHL. In o de o p edic he pa hological ole o he P o25Leu and Se 80Ile a ian s, i s we cons uc ed a mul iple sequence alignmen using e olu iona y dis an species. I has been shown p e iously ha he addi ion o mo e dis- an ly ela ed sequences is essen ial o a mo e accu a e p edic ion (20). Howe e , in ou case his app oach e ealed ha bo h a ian s migh be neu al, which is in con as wi h ea lie clinical indings sugges ing he dis- ease causing ole o he Se 80Ile bu no o he P o25Leu. The Se 80Ile has been p e iously epo ed only in one VHL pa ien wi hou clea cell enal ca cinoma [23], bu se e al o he missense mu a ions a codon 80 ha e been desc ibed. The Se 80Gly mu a ion has been de ec ed in a 12-y -old pa ien wi h bila e al pheoch omocy omas and mul iple congeni al mal o ma ions [24] and in a pa ien wi h pheoch omocy oma [25]. The Se 80Asn mu a ion was iden i ied in a Slo akian amily wi h VHL ype 1 phe- no ype including panc ea ic isle umo in one a ec ed indi idual [26]. In addi ion, he pa hogenic ole o Se 80A g [27,28] and Se 80Asn mu a ions has been also documen ed in VHL pa ien s [25]. Ou s udy shows o he i s ime ha he Se 80Ile mu a ion is associa ed wi h bila e al pheoch omocy oma p esen ed as a i s mani es- a ion o he disease, which poin s ou ha VHL pa ien s ha bo ing his mu a ion equi e ca e ul sc eening o phe- och omocy oma. The associa ion be ween a ious missense mu a ions a codon 80 o he VHL gene and VHL disease in p e ious s udies and ou s is no unexpec ed, since ou e olu iona y alignmen analysis showed ha in his posi ion he Se is a highly conse ed amino acid esidue among di e en species. Mo eo e , compu a ional p o ein modeling o a change o Se o Ile a posi ion 80 indica ed po en ially impo an consequences on h ee-dimensional s uc u e o he pVHL. Se 80 is a pola amino acid loca ed in he hyd ophobic co e o he β-domain close o he α-β in e - ace o pVHL and ElonginC in e ac ion. Acco ding o ou p o ein modeling, he change o Se o a la ge and non- Alignmen analysis o he VHL p o ein om Mus musculus (ENSMUSG00000033933), Ra us no egicus (ENSRNOG00000010258), Canis amilia is (ENSCAFG00000005149), Homo sapiens (ENST00000256474), Gallus gallus (ENSGALG000000013678), D osophila melanogas e (CG13221_CG13221-RA), Xenopus opicalis (ENSXET00000001448) and Taki ugu ub ipes (SINFRUG00000121189)Figu e 2 Alignmen analysis o he VHL p o ein om Mus musculus (ENSMUSG00000033933), Ra us no egicus (ENSRNOG00000010258), Canis amilia is (ENSCAFG00000005149), Homo sapiens (ENST00000256474), Gallus gallus (ENSGALG000000013678), D osophila melanogas e (CG13221_CG13221-RA), Xenopus opicalis (ENSXET00000001448) and Taki ugu ub ipes (SINFRUG00000121189). BMC Medical Gene ics 2008, 9:29 h p://www.biomedcen al.com/1471-2350/9/29 Page 6 o 8 (page numbe no o ci a ion pu poses) pola Ile a amino acid posi ion 80 esul s in a complex ea angemen o he h ee-dimensional s uc u e o he p o ein binding in e ace, which dis u bs HIF1α and ib onec in binding [29]. Mille and co-wo ke s ha e al eady demons a ed ha VHL gene mu a ions wi hin L1 and L7 loops dis up he dynamic coupling o he pVHL and HIF-1α and ha his may be an impo an mecha- nism o he de elopmen o umo s [30]. O he s udies using h ee-dimensional modeling and a compu e algo- i hm FOLDEF p o ided a quan i a i e es ima ion o he impo ance o he in e ac ions con ibu ing o he s abil- i y o p o eins and p o ein complexes [31]. In he case o he Se 80Ile, he ee ene gy di e ence be ween he wild ype and mu an p o eins was 12.2 kcal/mol, which is highe han he p e iously es ablished cu -o alue o he de elopmen o clea -cell enal ca cinoma o pheoch o- mocy oma [32]. Al hough mu a ions a ec ing he i s 53 codons o he VHL gene ha e no e ec on he s uc u e o he sho e VHL p o ein, a ew mu a ions a codons 25, 38, 46 and 52 ha e been conside ed o exe a pa hogenic ole. The P o25Leu and Se 38P o ha e been iden i ied in pa ien s wi h pheoch omocy omas, whe eas he Glu46S op and Glu52Lys ha e been associa ed wi h VHL disease [32,27]. Howe e , Ro hbe g e al. epo ed a VHL pa ien wi h wo VHL gene mu a ions, P o25Leu and P o86A g, in whom he P o86A g was conside ed mo e likely o be pa hogenic han he P o25Leu based on an allelic equency o 0.5% o he la e a ian in anonymized DNA samples [14]. This P o25Leu a ian was de ec ed in o he heal hy VHL Th ee-dimensional compu a ional modeling o he Se 80 and Ile80 esidues using CPK diag am (uppe pa ) and schema ic ep- esen a ion o hyd ogen-bonds be ween esidues Gly104 and Asn78, be ween A g79 and Ile151, be ween Se 80 and Ile151, beween Se 80 and P o103, be ween Se 80 and Leu153, and be ween P o103 and A g82 in wild ype pVHL and he conse-quences o he Se o Ile change a amino acid posi ion 80 (lowe pa )Figu e 3 Th ee-dimensional compu a ional modeling o he Se 80 and Ile80 esidues using CPK diag am (uppe pa ) and schema ic ep esen a ion o hyd ogen-bonds be ween esidues Gly104 and Asn78, be ween A g79 and Ile151, be ween Se 80 and Ile151, beween Se 80 and P o103, be ween Se 80 and Leu153, and be ween P o103 and A g82 in wild ype pVHL and he consequences o he Se o Ile change a amino acid posi ion 80 (lowe pa ). All igu es we e gene a ed wi h Swiss-PdbViewe in combina ion wi h POV-Ray. BMC Medical Gene ics 2008, 9:29 h p://www.biomedcen al.com/1471-2350/9/29 Page 7 o 8 (page numbe no o ci a ion pu poses) amily membe s om Poland and No h Ame ica [33,14]. In ag eemen wi h hese obse a ions, he P o25Leu ca - ie in ou amily had no mani es a ions o he disease, sugges ing ha his a ian does no ep esen a disease- causing mu a ion. Conclusion The Se 80Ile mu a ion o he hl gene in a la ge Hunga ian kind ed was ound o be associa ed wi h VHL ype 2 p e- sen ing wi h bo h pheoch omocy oma and enal cell can- ce . The e o e, pa ien s wi h his mu a ion equi e ca e ul su eillance, including sea ch o pheoch omocy oma. The P o25Leu a ian in his amily apea ed o be neu al, bu i s co-exis ence made gene ic counseling di icul . Abb e ia ions VHL: on Hippel-lindau disease; pVHL: on Hippel- Lindau p o ein; HIF: hypoxia inducible ac o . 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 da a: AP, KB, PG, FF pe o med he molec- ula biological analysis, AP, FF and IL pa icipa ed in alignmen analysis and in h ee dimensional p o ein modeling, MT and KR ob ained he clinical da a. Analysis and in e p e a ion o da a: AP, FF, IL, MT and KR. D a ing o he manusc ip : AP, KB, PG, MT, IL and KR. C i ical e ision o he manusc ip o impo an in ellec ual con- en : AP, IL, MT and KR. Adminis a i e, echnical, o ma e ial suppo : AP, PG, KB, FF, IL, MT and KR. S udy supe ision: KR. Acknowledgemen s W i en consen o publica ion was ob ained om he pa ien o hei el- a i e. Suppo ed by g an s om he Hunga ian Minis y o Public Heal h (EET 090/2006) Re e ences 1. La i F, To y K, Gna a J, Yao M, Duh FM, O cu ML, S ackhouse T, Kuzmin I, Modi W, Geil L, e al.: Iden i ica ion o he on Hippel- Lindau disease umo supp esso gene. Science 1993, 260(5112):1317-1320. 2. F ied ich CA: Geno ype-pheno ype co ela ion in on Hippel- Lindau synd ome. 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Publish wi h BioMed Cen al and e e y scien is can ead you wo k ee o cha ge "BioMed Cen al will be he mos signi ican de elopmen o dissemina ing he esul s o biomedical esea ch in ou li e ime." Si Paul Nu se, Cance Resea ch UK You esea ch pape s will be: a ailable ee o cha ge o he en i e biomedical communi y pee e iewed and published immedia ely upon accep ance ci ed in PubMed and a chi ed on PubMed Cen al you s — you keep he copy igh Submi you manusc ip he e: h p://www.biomedcen al.com/in o/publishing_ad .asp BioMedcen al BMC Medical Gene ics 2008, 9:29 h p://www.biomedcen al.com/1471-2350/9/29 Page 8 o 8 (page numbe no o ci a ion pu poses) 27. Doll us H, Massin P, Taupin P, Neme h C, Ama a S, Gi aud S, Be oud C, Du eau P, Gaud ic A, Landais P, e al.: Re inal hemangioblas - oma in on Hippel-Lindau disease: a clinical and molecula s udy. In es Oph h Vis Sci 2002, 43(9):3067-3074. 28. 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