MEDICAL INTELLIGENCE UNIT Molecular Mechanisms of Fanconi Anemia Shamim I. Ahmad, B.Sc, M.Sc, Ph.D. Schoolof BiomedicalandNaturalSciences NottinghamTrentUniversity Nottingham,England,U. K. Sandra H.Kirk, B.Sc.(Hons.), Ph.D. Schoolof BiomedicalandNaturalSciences NottinghamTrentUniversity Nottingham,England,U. K. LANDES B I O S CI ENCE /EUREKAH.COMSPRINGERS CIENCE+BUS INES S ME D I A GEOR GETOWN,TEXASNEWY O R K,NEWYORK U. S. A.U. S. A. MOLECULAR MECHANISMS OF FANCONI ANEMIA MedicalI ntelligenceUnit Landes Bioscience /Eurekah.com Springer Science+BusinessMedia, I nc. I SBN:0-387-31972-7Printedonacid-freepaper. Copyright2006Eurekah. comandSpringerScience+BusinessMedia,I nc. 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F36M652006 6l 6. 1' 52071- dc22 2005037313 CO NTENTS Prefacevii 1.ClinicalFeatures ofFanconi Anaemia1 A.MalcolmR.Taylor G eneticsofFanconiAnaemia2 ClinicalFeaturesof FanconiAnaemia3 FanconiAnaemiaandNijmegenBreakageSyndrome5 Confirmationof theDiagnosisinFA Patients6 R elationshipofComplementationG rouptoClinicalFeatures7 EvidenceforModifyingMutations10 2.TheGeneticBasis ofFanconi Anemia13 Grover C. Bagby, Jr. G eneticH eterogeneity13 TheiMA^C G enes14 FA ProteinComplexes21 3.TheFANCAGeneand Its Products28 LauraS.Haneline FANCAGenc28 FANCAProtein29 FANCAFunction30 AcquiredAMLandFANCADefects32 4.TheFANCCGeneand Its Products36 Susan M.Gordon andManuelBuchwald CloningandCharacteristicsof theFANCCGene36 MammalianH omologsofFANCC37 FANCCGeneMutations39 Expressionof theFANCCG eneProducts40 FANCCProteinExpressionandStability41 FANCCSubcellularLocalization41 FANCCintheCellularR esponsetoI CLI nducers42 Apoptosis43 FANCCLossofFunction43 FANCCandCytokineSignalling45 FANCCandO xidativeStress47 5.TheFANCB,E,FandG Genesand TheirProducts54 Filippo Rosselli FANCB55 FANCC56 FANCF57 FANCE57 6.FANCD1/B RCA2and FANCD261 Gary M.Kupfer TheFA-DComplementationG roup61 7.TheFANCGenomeS urveillanceComplex67 TakayukiYamashita H istoricalO verview67 Structureof theFA CoreComplex69 FunctionsoftheFA CoreComplex70 Perspectives71 8.O therProteinsand TheirInteractions withFA GeneProducts74 Tetsuya Otsukiand JohnsonM.Liu FANCC-BindingProteins7A FANCA-BindingProteins75 FA ProteinComplexandH umanaSpectrinI I78 9.Fanconi Anaemiaand O xidativeS tress: Cellular andClinicalPhenotypes82 GiovanniPagano andShamimI.Ahmad 10.Therapy forFanconi Anemia92 MadeleineCarreau Androgens92 H ematopoieticG rowthFactors92 BMTransplants93 G eneTherapy96 ProspectsforTherapy98 11.Mutational Analysesof Fanconi AnemiaGenes in JapanesePatients103 AkiraTachibana Patients,CellCultureandMutationAnalysis103 SequenceVariationsintheFANCAG ene104 SequenceVariationsintheFANCGG ene108 Mutationsoftheir^4A^CC G eneI l l MutationsofO therFA G enesI l l CharacteristicsandG eneticBasis of JapaneseFAPatientsI l l Index115 EDITO RSS hamimI.Ahmad S choolof B iomedicaland NaturalS ciences NottinghamTrentUniversity Nottingham,England,U.K. Email:shamim.ahmad@ntu.ac.uk Preface, Chapter9 S andra H.Kirk S choolof B iomedicaland NaturalS ciences NottinghamTrentUniversity Nottingham,England,U.K. Email:sandra.kirk@ntu.ac.uk Preface CONTRIBUTORSGrover C.B^by,Jr. O H SUCancerI nstitute DepartmentsofMedicine and MolecularandMedicalGenetics O regonH ealthandScienceUniversity Portland,O regon,U.S.A. Email:grover@ohsu.edu Chapter 2 ManuelBuchwald Departmentof Molecular and MedicalGenetics UniversityofToronto Toronto,O ntario,Canada Chapter 4 MadeleineCarreau DepartmentPediatrie Universite Laval Unitede Recherche enG enetique H umaineetMoleculaire CH UQ,PavilionSt-Francois d'Assise Quebec, Quebec,Canada Email:madeleine.carreau@crsfa.ulaval.ca Chapter 10 SusanM.G ordon PrograminGenetics andG enomicBiology ResearchI nstitute H ospitalforSickChildren Toronto,O ntario,Canada Email:sgordon@sickkids.ca Chapter 4 FumioH anaoka G raduateSchool of FrontierBiosciences SO R ST JapanScience andTechnology Corporation Suita,Osaka, Japan Preface Laura S.H aneline Departmentof Pediatrics H ermanB. WellsCenter forPediatricResearch I ndianapolis,I ndiana,U. S. A. Email:lhanelin@iupui. edu Chapter 3 G ary M.Kupfer DepartmentofMicrobiology Departmentof Pediatrics DivisionofPediatric H ematology-O ncology Universityof Virginia Charlottesville, Virginia,U.S.A. Email:gk9e@Virginia.edu Chapter 6 JohnsonM.Liu SchneiderChildren'sH ospital PediatricH ematology/O ncology andStem CellTransplantation New H ydePark,New York,U.S.A. Email:jliu3@NSH S. edu Chapter 8 TetsuyaO tsuki ClinicalSciencePlanning andDevelopment Banyu(Merck-Japan)Pharmaceutical Company Tokyo, Japan Email:tetsuya_otsuki@merck. com Chapter 8 G iovanniPagano Centre forResearch I nnovationand TechnologyTransfer inO ncology andLifeSciences Mercogliano(AV), I taly Email:gbpagano@tin. it Chapter 9 FilippoRosselli Laboratoryof G eneticStability andCancer UPR 2169, CNR S Gustave Roussy I nstitutePR2 Villejuif,France Email:Rosselli@igr.fr Chapter 5 AkiraTachibana R adiationBiologyCenter KyotoUniversity Kyoto, Japan Email: atachibana@house. rbc. kyoto_u. ac. jp Chapter 11 A. MalcolmR.Taylor CR -UKI nstituteforCancerStudies University ofBirmingham Birmingham,England,U. K. Email:A. M. R . Taylor@bham. ac. uk Chapter 1 TakayukiYamashita Divisionof G eneticDiagnosis I nstitute of MedicalScience University of Tokyo Tokyo, Japan Email:y-taka@showa.gunma-u.ac.jp Chapter 7 PREFACE Fanconi anemia(FA) is a rare largely autosomal recessive genetic disor-der(onecomplementationgroupbeing X -linked)thatwasfirstrecognized almost40yearsago^ as a causeof juvenileleukemia.O therphenotypesin-cludebonemarrowfailureleadingtoaplasticanemia,growthretardation, congenital malformationsof renal, cardiac, skeletal and skin structures,pan-cytopeniaandpronouncedcancerpredisposition. I nterestinglyFAsharesanumberofclinicalandmolecularfeatures with a variety of other syndromes, including Seckel syndrome^ andNijmegen breakagesyndrome,^andfiirtherinvestigationofallthreeconditionswill provideinsightsintothemolecularmechanismsinvolvedineachoneand where theymayinteract. I ntherecentpastmuchefforthasgoneintounderstandingthemo-lecular pathogenesis of FA in terms of enhanced susceptibility to DNAdam-aging agents. Results of these studies have yieldedexciting informationona multiplicityofhithertounknownprotein-proteininteractionsinvolvedin activitiesfromcontrolof redox state andapoptosistorepair of DNAstrand breaks. ^^ W orkingwiththeleadingresearchersandcliniciansinthefield,this bookhasbeenproducedtoprovideacomprehensivetreatiseonFA.This covers indetail what is knownof the12 complementationgroupsidentified todate. TheseincludeFANCA,-B(X -linked,localizedatX p22. 31),' '-C, - Dl ,-D2,-E, -F, -G ,-I , -J,-L andM. Thereis clear variationin the clinical featuresexhibitedby sufferersin differentcomplementationgroups,andevenbetweenthosewithdifferent mutationsofthesamegene.ThisisexploredindetailinChapter1and expandeduponintheintroductiontoChapter5. Thelinkbetweenmuta-tionandphenotypemayultimatelyhelppickapartthecomplexcellular actionsoftheFANCproteins.Attheotherendofthespectrumandthe book,Chapter10detailsthebesttreatmentsas currentlyperceivedforFA. W hilstthefutureholdsoutthepossibilityofgenetherapyorproteinre-placement,thetreatmentof choiceatpresentis still stemcell orbonemar-row transplantationfroma matchedsibling.^'^ Chapter 3 introduces the FANCgenes identifiedto date and their prod-ucts.I tis apparentthatFANCA,-B,-C,-E,-F, -Gand-Lforma corecom-plex within the nucleus of cells leading to monoubiquitinationof FANCD2. ATRcheckpointkinaseandR PAlarerequiredforefficientFANCD2 monoubiquitination. ^Thenatureandroles of this complex are thesubjects ofChapter7.FANCDlandFANCJappeartoactdownstream,beingac-tively recruitedalong with BR CAland R AD51to chromatinat specificfoci forthe repairof DNAdamage. Theprecise role of FANCIhas notyetbeen determinedbutitseemstoactatastagebetweencorecomplexformation andFANCD2ubiquitination. ^^Theinvolvementofbreastcancersusceptibility andFA genes in this pathway is an area of active research. I t is evident thatFA-D1 patientsarepredisposedtobreastcancer/^althoughmembersoftheotherFA complementationgroups do not show a high frequencyof this malignancy. ^^ This wouldseemtoindicatethattheBR CAproteinsmayexerttheirbreastcancer preventativefunctionindependentlyofFANCD2ubiquitination(althoughsee below).BR CAlandBR CA2areknowntobeindependentlyinvolvedinother activities including regulation of differentiation,and it may be that thedisruption of theseimpactsonbreastcancerdevelopment. ^ I nadditiontotheircentralroleinDNArepairtheFAproteinsareinti-matelyinvolvedinarangeofothercellularfunctionsthroughtheirindividual interactionswithotherproteins. Thisconceptis introducedinChapter2where proteinsinteracting withFANCA,FANCCandFANCGare introduced.Several of these interactionsare withproteinsinvolvedinmaintenanceof redoxbalance. I nChapter9,PaganoandAhmad,longtermproponentsoftheoxidativestress theoryof FA, expanduponthe waysinwhichinterruptionsinFA-redoxprotein associations couldresult inthe phenotypicabnormalitiesobservedinFA, provid-inganalternativemodeltothemorewidelyacceptedDNArepairdeficiency. Abnormalitiesin mitochondrialfunctionin FA furthersupporta role of oxidative stress."^I tislikelythatitisacombinationofadeficiencyinredoxcontroland ineffectiveDNArepair whichgive the complexclinical characteristicsof this dis-order: many of the congenital abnormalitiesarising due to the former,and predis-position to malignancy to the latter. I t is highly probable, however, that the debate overtherelativecontributionsofthesetwophenomenawillcontinueforsome time. MutationsinFANCA,-C and-Gare the mostprevalentandtheindividual genesandtheirproductsareconsideredindetailinChapters3-5.I nterestingly otherproteins whichFANCA, -C and-Ginteract with are knownor suggestedto be involved in chromatinremodeling(e. g. , FAZF^^ and BR G l),^^ andFANCCin particularappearsto have a role in protectionagainst cytokine-inducedapoptosis inhaematopoieticcells. ThesephenomenaarediscussedindetailinChapters4 and8andevidenceisbeginningtoappeartosuggestthatdifferentstructural domainwithinFANCCareresponsibleforitsdifferentactivities.FANCGcon-tains sequence motifstypical of a protein involved in protein-proteininteractions, andit is essential forthe functionalinteractionbetweenFANCCandFANCA.I n turnits ability tointeractwithFANCArequiresthe presenceof FANCB,-Cand -F, illustrating furtherthe intricate interplay of these proteins which is exploredin detailinChapter5. Chapter6considersthenaturesofFANCDland-D2proteins. Theexcit-ingdiscoverythatFANCDlisinfactthebreastcancersusceptibilitygene productBR CA2,andthatubiquitinatedFANCD2interactswithBR CAlhave hugely expandedthe research effortsin this area. ThenuclearFA complexappears toexertitseffectsinatwoprongedapproach:byubiquitinationofFANCD2 which in turnrecruits BR CAlto the nuclearfoci formedat DNArepair sites, and via interactions(possibly throughFANCGassociation) with BR CA2 andR AD51, theseproteinsalsobeingrecruitedtodamagefocialthoughseeminglyindepen-dentlyofBR CAl. ^^Thedetailoftheseinteractionsandtheirrolesindamage repairareunderintenseinvestigation,particularlyas BR CAlrecruitmentseems tobe implicatedina range of DNArepairmechanisms. ^^ Thefinalchapter(Chapter12)is devotedtoanindepthdiscussionofthe mutationsfoundina geographicallyandgenetically isolated Japanese FA popula-tion. I nterestingly this group shows a preponderance of mutationsin FANCA as is observedin the W est,butalso shows a higherfrequencyof FANCGinvolvement, confirmingthecentralrole of FANCAandtheFANCA-FANCC-FANCGcom-plex inprotectionagainstFA. Arecent Tunisianstudyof 41familieshasrevealedthat92%belongtothe FANCA complementationgroup. ^ O thershave identifiedSpanishG ypsiesasthe ethnicgroupwiththeworld shighestprevalenceofFAthecarrierfrequency being1/64-1/70. ^AffectedindividualsarehomozygousforaspecificFANCA mutation(295C>T)leading to FANCA truncation. This mutation was notfound in other G ypsy patients fromH ungary, G ermany, Slovakia and I reland. I n a South-ernAfricanpopulation(SouthAfrica,Swaziland,MozambiqueandMalawi)a particulardeletionmutationinFANCG(c. 637643delTAACCG CC)hasbeen reported in 82% of FA patients. Birth incidence of FA in this populationis greater than1/40,000whichisaboveaverageingeneralpopulations.Thesestudiesare clearly indicative of foundermutationin the Spanishgypsy andSouthernAfrican populations. ThereisnoevidencethatinterestinFAiswaning,andthisbookshould provide boththe experts andnovice researchers in the field withan excellentover-view of thecurrentstatusof researchandpointersto fixture researchgoals. Not eAddedi nProof I n a recent study Meetei et al have identifieda new complementationgroup, FANCM.Theprotein,FAAp250,is a partoftheFA proteincomplexinvolving BR CAlandBR CA2andhassequencesimilaritytootherknownDNArepair proteins,includingH efinarchaea,MPH linyeastandER CC4orX PFinhu-man.FANCMisessentialformonoubi qui t i nat i onofFANCD2andis hyperphosphorylatedwhenDNAisdamaged. ShamimLAhmadyFutnioHanaokaandSandraH.Kirk References 1.AndreassenPR ,D' AndreaAD,TaniguchiT.ATRcouplesFANCD2 monoubiquitinationtotheDNA-damageresponseG enesDev2004; 18:1958-1963. 2.BouchlakaC, AbdelhakS, DellagiK. Molecularstudy of Fanconianemia in Tunisia. TunisiaMed2004;82:4023-410 3.CallenE,Cassado JA, TischkowitzMDetal. Acommonfoundermuta-tionin FANCA underlies the world highestprevalence of Fanconianemia ingypsy familiesinSpain.Blood2005;105:1946-1949. 4.Clarke AA,G ibsonFM,Scott Jetal.Fanconianemiacell lines show dis-tinctmechanismsofcelldeathinresponsetomitomycinCoragonistic anti-Fasantibodies.H aematologica2004;89:11-20 5.FanconiG .Familialconstitutionalpanmyelocytopathy,Fanconi s anemia (FA).IClinicalaspects. SeminH ematol1967;4:233 6.G ennery AR , Slatter MA, Bhattacharya A et al. Theclinical andbiological overlapbetweenNijmegenbreakage syndromeandFanconianemia.Clin I mmunol2004;113:214-219. 7.G rewalSS, Kahn JP, McMillanMLetal. Successfulhaematopoieticstem celltransplantationforFanconianemiafromanunaffectedH LA-geno-type-identicalsiblingselectedusingpreimplantationgeneticdiagnosis. Blood2004;103:1147-1151 8.G uardiolaP, Socie G ,Li X et al. Acute graftversus-hostdisease inpatients withFanconianemiaoracquiredaplasticanemiaundergoingbonemar-rowtransplantationfromH LA-identicalsibling donors:Riskfactorsand influenceonoutcome.Blood2003;103:73-77 9.KubistaM,R osnerM,Miloloza A et al. BR CAlanddifferentiation.Mut Res 2002;512:165-172. 10.LevitusM,R ooimansMA,SteltenpoolJetal.H eterogeneityinFanconi anemi a:evi denceoftwonewgenet i csubt ypes.Blood2004; 103:2498-2503. 11.Meetei AR ,LevitusM,X ue Y etal. X -linkedinheritanceofFanconiane-miacomplementationgroupB. NatG enet2004;36:1219-1224. 12.O tsukiT,FurukawaY,I kedaKetal.Fanconianemiaprotein,FANCA associateswithBR G l ,acomponentofthehumanSW l / SNFcomplex. H umMolG enet2001;10:2651-2660. 13.R enter TY, Medhurst AL, WaisfiszQe tal. Yeast-two hybridscreensimply involvementofFanconianemiaproteinsintranscriptionregulation,cell signaling, oxidative, metabolism, and cellular transport. Exp Cell Res 2003; 289:211-221. 14.Seal S, BarfootR, Jayatilake Het al. Breast cancer susceptibilitycollabora-tion.Evaluationof Fanconianemiagenes infamilialbreastcancerpredis-position.CancerRes2003;63:8596-8599. 15.ThompsonLH ,HinzJM,YamadaNAetal.HowFanconianemiaproteins promotethe fourRS: repUcation,recombination,repair andrecovery. Environ Molec Mutagen 2005; 45:128-142 16. WagnerJE, TolarJ,LevranOetal.GermlinemutationsinBRCA2:shared genetic susceptibiUtytobreastcancer,early onsetleukemia,andFanconiane-mia. Blood 2004;103:3226-3229. 17. W angX,AndreassenPR ,D'AndreaAD.Functionalinteractionof monoubiquitinatedFANCD2andBRCA2/FANCD1inchromatin.Mol Cell Biol 2004; 24:5850-5862. 18.Meetei AR,MedhurstAL,LingCetal. A humanorthologofarchaealDNA repairproteinHef is defectiveinFanconianemiacomplementationgroupM. NatGenet 2005; 37:958-963. Acknowledgments W e wouldlike toacknowledgethecontinuingsupport of ProfessorFumioH anaokaof theUniversity ofO saka forhis encouragementinourinterestinFanconianemia. CH APTER1 ClinicalFeatures of Fanconi Anaemia A. Malcolm R. Taylor* Abstract Fanconianaemiaisanautosomalrecessivedisorderinwhichpatientsdevelopbone marrow failureand aplastic anaemia butthis can occur at widely differingages fromthe first year to age 12 years or more. This means that oftenthe diagnosis is made beforethe onset of any haematologicalabnormality is apparent. Many, but not all, FA patients have quite severe congenitalabnormalitiesand so the unusual sensitivity of FA peripheralbloodlympho-cytestoDNAcrosslinkingagentshasbeenanimportantpartof thediagnosisinFA.FA patients, however, may have neither bone marrow failure nor congenital abnormalities, when a diagnosis ofFAis suspectedandthis has led tooccasionalconfiisionwithNijmegenBreakage syndrome where there is also unusual sensitivity to DNAcrosslinking agents. I ncontrastcells fromFA patients, withsome rare exceptions, donotshow anincreasedsensitivity toionising radiation. The absence of particularFANC proteinscan result in a more severe phenotype;for example the predispositionto boththe presence of congenital abnormalities withearly diagnosis in patients with FANCD2mutations and very early onset of myeloid or lymphoid leukaemia as well as carcinomasin the case of patients with FANCDlmutations. The pathogenesisof bone marrowfailureandcongenitalabnormalitiesisnotunderstoodinthecontextofthelossof particularFANCproteins. I naddition,thenumberof genes identifiedis fairlylarge andtheir exact roles in the FApathways have not yet been fiiUy worked out. Although the cellular targets forthese differentFA proteinsand the FA pathway have not yet been elucidatedthereappears to be a role forthe FA proteinsin homology directedrepair of DNAdouble strandbreaks. Introduction The interest in the autosomal recessive disorders Fanconi anaemia(FA),Ataxia-telangiectasia (A-T),andBloomsyndrome(BS) was originallydrivenby theknowledgethateachgroupof patientshasastrongpredispositiontocancer,withadifferentspectrumoftumoursineach disorder.^I naddition,ithadbeenshownthatspontaneouslyoccurringchromosomeabnor-malitiesoccurredinFA, A-TandBS andthey became knownas the ChromosomeI nstability Syndromes.Althoughthistitleperhapsinferredsomethingsimilaraboutthechromosome abnormality between themit was,in fact,quite differentin each disorder. Thenotionthatthe chromosomeabnormalitywas relatedtothetumourpredispositionwas clearlysuspected although the mechanism for this, in each case, was not then understood. Patients withanother disorder.X erodermapigmentosum(XP)whohavea predispositiontoskincancerwerefirst showntobeunusuallysensitive toanenvironmentalagent,ultravioletlight(UV).'^ This was demonstratedtobetheresultof a defectinDNAexcisionrepairofUVinducedpyrimidine dimers.Thereafter,FAandA-TpatientswereshowntobeunusuallysensitivetoDNA *A. MalcolmR. TaylorCR-UKInstitute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, BirminghamB15 2TT, England, U.K.Email: A.M.R.Taylor@bham.ac.uk Molecular Mechanisms of Fanconi Anemia^ editedby ShamimI . Ahmadand SandraH .Kirk. 2006Eurekah. comandSpringerScience+BusinessMedia. Molecular Mechanisms of Fanconi Anemia crosslinking agents, mitomycin C(MMC)and diepoxybutane(DEB)^ and ionising radiation respectively, with the inference that they also would have a defect in some formof DNA repair. Subsequentlyanothergroupofpatients,withNijmegenBreakageSyndrome(NBS),^were described with spontaneouschromosome abnormalities like those of A-T, an unusual sensitiv-itytoionisingradiationandapredispositiontolymphoidtumours,againlikeA-T.The variousgenesforX P(XPA-Gandvariant)andalsothoseforA-T{ATMandhMREll),^'^^ NBS(NBSiy^'^^andBS (BLM)were identifiedandthe molecular pathology of each of these disorders has been investigated intensively since. I n contrast to A-T and BS, but in this respect more similarto XP, several geneshave beenidentifiedthat, whenmutated,give rise toFA. A relationship has been demonstrated between FA, A-T^^ and disorders where either hMREllor NBSlare mutated.Therefore, interestingly, there has been some convergence of these disorders oratleastthebiochemicalpathwaysunderlyingthemandthey havenowbeenreferredto as genomic instability syndromes.^^ GeneticsofFanconiAnaemia There are 12 differentcomplementation groups for Fanconi anaemia of which nine,FANCA, FANCB, FANCQFANCD1{BRCA2),FANCD2,FANCE, FANCF, FANCG{XRCC9) FANCL and FANCMhsivebeen cloned. Genes for FANCI , FANCJ remain to be cloned. The FA genes are quite differentfromeachothershowing litde ornohomology. The proteins encoded by FANCA,FANCQFANCE, FANCF, FANCG{XRCC9) and FANCL forma nuclearcomplexthatacts as a ubiquitinligase withtheFANCL componentbeingthe catalytic subunit.The protein complex is required for the damage inducedmonoubiquitination of the downstreamFANCD2protein(see also the sectionbelow onconfirmationof the diag-nosis of FA). The monoubiquitinated(activated) form of FANCD2 colocalises with BR CAl, BRCA2(FANCDl)^^andNBS^^innuclearfociaspartoftheresponsetoDNAdamage. FANCI may also be part of this complex.Recendy, following an analysis of this same protein complex, Meetei et al^^ identifieda proteinthey termedFAAP95 encodedby a gene on the X chromosome.Theywereabletoshowthatknockdownofthisproteinresultedinreduced ubiquitinationof FANCD2.Further, they showed that FAAP95 mutations are the likely cause ofFA-B.FANCD2canbemonoubiquitinatedinbodiFANCD1/BR CA2andFA-J cells^^ indicatingthatbothFANCDlandthepredictedFANCJproteinactdownstreamintheFA pathway. AsurprisingandintriguingfindingwasthattheFA-Dlcomplementationgrouprestdts frombiallelicmutationofBRCA2,^^mutationof whichis associatedwithpredispositionto breast and other cancers. The BRCA2 protein has known functionsin homologousrecombina-tion repair.Although biallelic null mutations in mice are embryonic lethal FANCDlFanconi patientshave hypomorphicalleles resulting inexpressionof sometruncatedBRCA2with partialactivity(see below forcharacteristicsof FA-Dlpatients). SinceFA patients'cells areunusuallysensitivetoDNAcrosslinkingagentsitis presumed thata role of the FA proteincomplexis the resolutionof DNAcrosslinks. Interestingly,how-ever,relatively litde hasbeendoneonthisrepairpathway. Thetargetsof theFA pathwayare not presently known. Overall, the normal functionof the FA/BR CAl/2 pathway is assumed to be part of the cellular response to damage present at S phase as a consequence of DNA interstrand crosslinks.MonoubiquitinatedFANCD2isrequiredfortargetingBRCA2tochromatin. G iventheinvolvementof FANCDl(BR CA2)inhomologousrecombinationrepairthese results suggests that the FA protein complex, generally, is also involved in this pathway. I ndeed, bothupstreamFANCAandFANCGanddownstreamFANCD2proteinsappeartobe involvedinrepairof homologydirectedrepairofDNAdoublestrandbreak(DSB)andalso single strandannealing.^^ Clinical Features ofFanconiAnaemia Table1.Clinical features ofFanconi anaemia Present at birth Microcephaly Growthretardation Skin pigmentationabnormalities Congenitalabnormalities Upperlimb abnormalities(thumb and radialray abnormalities) Renal Urogenital Gastrointestinal Other Neurological Variable age of onset Bone marrowfailure Myelodysplasticsyndrome (MDS) Acute myeloidleukaemia (AML) Carcinomas AdaptedfromdatainDuckworth-Rysieckietal,^9M^,^^ Alter,1993;^^Faivreetal,2000;^^ Tischkowitzand Hodgson, 2003;^^Huber and Mathew, 2004'^'' ClinicalFeatures of Fanconi Anaemia(Table1) FA is a rare disorder with an incidence of approximately 3 cases per million,^^''^very similar to the birth frequencyof A-T.The median survival period is ^2A years.Since so many genes arenowknowntobeinvolvedinthedevelopmentofFAaconsiderabledegreeofclinical heterogeneitymightbepredicted,althoughthedegreeof heterogeneitymayalsodependon the frequencyof each genotype. Haematological Abnormalities This is the most serious andconsistentof the clinical featuresof FA.I nthe originalpub-licationbyFanconi(1927)theaplasticanaemiawasthemajorfindinginthereportonthe three affectedbrothers. I t would seem appropriate, thereforethat a sine qua non for the diagno-sis of FA wouldbe the presence of aplastic anaemia. Theproblemis thatthepancytopeniais not usually present at birth but only develops later, in most instances between the ages of 5 and 10 years with median age of onset at 7 years."^^ However, other features of FA, like the presence ofcongenitalabnormalities,maybeimmediatelyapparentanddiagnosisis frequentlymade beforethe onsetof theaplastic anaemia. Thiscanhave theadvantagethat,if thediagnosisis correcdymade,the eventualanaemiamay be bettermanaged.Exceptionally,pancytopeniais seenas early as neonatallifeand Landmannet al^^ reportedpancytopeniaat age 2 weeks ina FA patient without congenital abnormalities with the homozygous FANCG mutation1649delC. Bonemarrowfailureprecedesthedevelopmentofmyelodysplasticsyndrome(MDS)or acute myeloid leukaemia (AML) although occasionally patients can present with either of these. Drawing ondata fromthree sources, a literaturesurvey,the North AmericanSurvey of145 Fanconipatients^ ^ andtheI nternationalFanconiAnaemiaRegistry(IFAR)study.Alteret al^"^ suggestedthatthecumulativeincidenceofanyhaematologicalfindinginFA maybeas high as 90%, and bone marrow failure requiring therapy was -60%. This agrees with an earlier estimate by the I nternationalFanconi Anaemia Registry^^ that the actuarial risk of developing any haematopoietic abnormality by age 40 was 98%; the risk of pancytopenia was 84% by the age of 20 years and the risk of either MDSor AML was about50% by age 40 years (MDSand AMLwerenotconsideredseparately).Deathresultedfromhaematologicalcausesin80%of cases by age 40years. Therisk of bonemarrowfailurewas highestin patientswithabnormal Molecular Mechanisms of Fanconi Anemia Table 2.Malignant disease in Fanconi anaemia patients Leukaemias Acute myeloidleukaemia (AML) -5-10% risk Myelodysplaticsyndrome 5 % risk Acute T cellleukaemia (T-ALL) -rare; onlyreportedinFANCD1 (BRCA2) patients Carcinomas Liver tumours -may be related to treatment Head and neck tumours Oesophagus Vulva Embryonal tumours Wilms tumour-rare; onlyreportedinFANCD1(BRCA2)patients Brain tumours Medulloblastoma -rare; onlyreported inFANCD1(BRCA2)patients Astrocytoma -rare; onlyreportedin FANCD1 (BRCA2) patients Adapted from data in Offit et al, 2003;^^ Hirsch et al, 2004;^^ Wagner et al, 2004;^Alter, 2003;2S Alter et al, 2003^^ radii and a 5-point congenitalabnormality score. ^Conversely the greatest risk fordeveloping AMLora solidtumouroccurredinthelongestlivedpatients-those withthelowestrisk of bonemarrow failure. ^^ Approximately30% of FA patients studiedby Rosenberg et al^^ andKuderet alreceived abonemarrowtransplant(BMT).I nthe145patientsinthestudyofRosenbergetal44 patients underwentBMT, 37 for aplastic anaemia and 7 for MDS. The complications of BMT also carry a risk of death.^^'^^ Leukaemia in Fanconi Anaemia According to Alter et al^"^ the individual's risk of MDS was --5% with the evolution to AML not being inevitable. Their estimate was that,in addition,there was a 5-10%individual risk of AML in FA. SolidTumours(Table 2) The risk of solid tumoursincreases with age with the risk being 5-10%and cumulative risk of-30%by age 45 years,greater than for AML. Young adults with FA show an increased risk of head and neck squamouscell carcinomastumours, tumours of oesophagus, kidney(includ-ing embryonic W ilmstumoursand nephroblastoma see below), brain(mainlymedulloblas-toma),liver,vulvaandcervix. Theoccurrenceoflivertumoursmayberelatedtotreatment withandrogens(oxymethalone)forbonemarrowfailure.Somepatientshavealsodeveloped twotumoursand,morerarely,morethantwo.^TheconditioningregimenforBMTis also associated with risk of inductionof malignant disease. The risk of squamous cell carcinomas of the head,neck andoesophagus was reportedly higherinFA patients who hadreceived a bone marrowtransplantandoccurredat anearlier age thannontransplantedpatients. ^ Presenting Features of FA (Table 1) Small stature and the presence of one or more congenital abnormalities(principally micro-cephalyandabnormalitiesof skinpigmentation)intheabsence of aplasticanaemiais, there-fore, how many FA patients present. They show slower growth bothin utero, with lower birth weight, and afterbirth. This may be furthercomplicated by the presence of hormonaldeficien-cies.' T wothirds of patients will also show multiple congenital abnormalities. These include upperlimbabnormalities,especiallyabsentradiusandthumbanomalies,as wellasrenal Clinical Features ofFanconiAnaemia abnormalities(e. g. , horseshoekidney).Less commonlytheremay be abnormalitiesofthe urogenitalandgastrointestinalsystems,thelowerlimbs,cardiopulmonarydefects,deafness andneurologicalabnormalities^^'as well as more rare pathologies. ^ ThemajormethodforlaboratoryconfirmationofthepresenceofFA hasbeentousea peripheralbloodsampletodemonstratetheunusualchromosomalsensitivityofFA patients' cells to DNAcrosslinking agents(see ref.41). Cells fromall FA patients show increased sensi-tivity to DNA crosslinking agents although there is some variation between patients in the level ofinduceddamage.Thelevelofspontaneouslyoccurringchromosomedamageisvariable being raisedinlymphocytesfrommany butnotall FA patients. ^ Fanconi Anaemiaand NijmegenBreakageS yndrome Notall FA patients show the presence of congenitalskeletal, gastrointestinal,renal tractor central nervoussystem(CNS)abnormalities.I ndeeda study suggested thatas many as a third of patients fallintothis category.^ If a diagnosisof FA anaemia is suspectedat anearly age in this group of patients, the aplastic anaemia is unlikely to be present and the diagnosis relies on thepresenceofotherfeaturessuchas microcephaly,growthretardationandabnormalitiesof skinpigmentation. NBS patients also share some features withFA including microcephaly and skinpigmenta-tion changes(Table 3). Recently, a patient originally diagnosedat age 2 years with atypical FA wasshowntobehomozygousforanNBSlmutation{NBSl1089C>A;Y 363Xwhichis differentfromthecommon657del5Slavicmutation)andshowedtotalcellularlossof Nbs 1 protein. ^ 'The patient did not show any aplastic anaemia, but did show microcephaly, skinpigmentationabnormalities,thrombocytosis,mildlymphopaenia,recurrentupper respiratory tract infections with an increased level of DEB induced chromosome breakage and so wasclassedas havingFA.Thispatienthadtwosiblingswiththesamesymptomsanda diagnosisof FA wasagainmadeonthebasisof thesefeaturesanda positiveDEBtest. Their cousin, patient1,showed microcephaly, failureto thrive, recurrentchest infections,andcafi au lait spots. Later immunodeficiency was also diagnosed. She subsequendy developed a plasma cell tumouranddied. None of these patients haddevelopedan aplastic anaemiabuta second child of--4 years died fromcomplications followingan infectionand two patients aged^6and 3 years, respectively,are alive. Cells fromboth A-Tlike disorder(ATLD- mutatedforhMR El 1) and NBS patientshave also been shown to be unusually sensitive to DNA crosslinking agents, DEB and MMC. ^^ This observation,therefore,showed that increased sensitivity to either of these agents is notspecific forFA. AfurtherreportdescribedtwopatientsintworelatedfamilieswhereFA wasapossible diagnosisbutinfactthepatientswere showntohavethe NBSl1089C>A; Y363Xmutation andapparenttotalabsenceofNbs 1.Interestingly,thisisthesamemutationasthepatients described above.'The patients describedby G enneryet alwere small withmicrocephaly, repeated infections,immunodeficiencyand failureto thrive. Radial ray anomalies andaplastic anaemiawerenotpresent. TheauthorssuggestedthattheclinicalfeaturesoftheseNBS patientsappearedtobe particularlysevere comparedwithotherNBSpatients.I tis notclear how the NBSl1089C>A; Y363X homozygousmutationresults inthisparticularphenotype. Although fulllength Nbslprotein could not be demonstrated,it is unlikely to be totally absent as most NBS patients show some truncated Nbsland total absence of the protein is embryonic lethal in mice. I n conclusion, cells from these patients show that (i) increased sensitivity to DNA crosslinking agents like MMCandDEBis not specific toFA patientsand indeedit is commontopatients withFA,NBSandATLDas wellascellsfromsomeSeckelsyndromepatients(ii)inthe absence of congenitalabnormalities, there is some overlap of clinical featuresbetweenFA and NBS especially with regard to microcephaly and skin changes although the primaryimmimodefi-ciency of NBSis not seen in FA andaplastic anaemia associated withFA is notseenin NBS. Molecular Mechanisms of FanconiAnemia Table 3,ComparisonoffeaturesofFanconi anaemiaandNijmegenBrealiage syndrome Feature CLINICAL Mi crocephal y Growt hretardation Skinpigmentationabnormalities Typicalfacies Craniofacialfeatures Learningdifficulties Aplasticanaemia Primaryi mmunodefi ci ency Infections Myel oi dtumours Lymphoi dtumours Othertumours CongenitalAbnormal i ti es Radialrayabnormalities Renalabnormalities Urogenitalabnormalities Deafness CYTOGENETICSOFPERIPHERALLYMPHOCYTES Spontaneousrandombreakage Translocationst(7;14)etc CELLULAR Increasedsensitivityto DNAcrossl i nki ngagents Ionisingradiation Adapted fromdata in Hi el et al , 2 0 0 0 /Alter,1993; ^2003; ^^HuberandMathew,2004^^ FanconiAnaemia yes yes yes yes(some) yes(some) mi nori ty yes(lateronset) no no yes rare yes(carcinomas) yes yes yes yes yes yes(some) no yes no(rareexceptions) NijmegenBS yes yes yes yes yes(all) majority no yes yes no yes yes(nocarcinomas) mi nororrare no no no no no(low) yes yes yes(all) Faivreetal , 2000; ^^ Ti schkowi tzand Hodgson, Confirmationof theDi s ^os i sinFA Patients ThefindingthatincreasedsensitivitytoeitherMMCorDEBisnotspecificforFA has influencedthe methods of confirmationof the diagnosis. However, where there is both aplastic anaemiaandcongenitalabnormalities,FA is a likely diagnosis.I nthisgroupof patients,the presence of increased chromosome breakage, following exposure of blood to DNA crosslinking agents,shouldconfirmthediagnosis. ThemajorityofFApatientsareincomplementation groupsFANCA(^70%),FANCC(-10%)andFANCG(-10%).Complementationanalysis by infectionwithretroviruscontaininga cDNAfroma FA gene^ will identifytheparticular FANCgeneandsubsequentDNAsequencingwillidentifythegenemutationinthevast majorityof these cases. An alternative method for establishing the likelihood that a patient has FA is to examine the ability of the patients primary lymphocytes to monoubiquitinatethe FANCD2 protein.'' '^ TheFANCA,FANCC,FANCE,FANCF,FANCGandFANCLproteinsinteractwitheach otherandthecomplexisrequiredforthemonoubiquitinationofthedownstreamprotein FANCD2.ProteinextractsofnormalcellsimmunoblottedforFANCD2showtwobands, with the larger formbeing the ubiquitinatedFANCD2. Cells fromFA patients withmutations in FANCA, FANCC, FANCE, FANCF, FANCG or FANCL express only the nonubiquitinated smallerisoformofFANCD2.Theabsenceoftheubiquitinatedbandincellextractfroma Clinical Features ofFanconiAnaemia patientwillindicateFA causedby mutationinoneofthese genes. Thisof course willbethe majorityof FA patients. Complementationanalysis, followedby sequencing, will identifythe FANC gene mutation.Some cautionis requiredas there are other FANC genes. The very rare FANCD1(BRCA2)mutationencodesa proteinthatfunctionsdownstreamof FANCD2and so ubiquitinationof FANCD2in cells fromsucha patient will give a falsenegativeresult. I naddition,mosaicisminbloodcellsmayalsoleadtoa falsenegativeif therevertant normalpopulationis largeenough.Mosaicismforcells witheitherbiallelicorsingleFANC mutationsaffectsaminorityofFAindividualsandseenasaclearunusualsensitivitytothe chromosomedamagingeffectsofcrosslinkingagentsinsomebloodlymphocytesbutnotin others. Recently, Soulier et al^ reported the presence of mosaicism as shown by the presence of alightmonoubiquitinatedFANCD2bandinFA-Aperipheralbloodcells. W hatisthe mechanismforthe reversion of phenotype? Reversion of a mutationin cells heterozygous for a mutationmay occur through gene conversion. I n the case of FA, where both alleles are mutant, onemechanism,forexample,wasdescribedinwhichasinglebasedeletion(resultingina frameshiftmutation)wascompensatedbytwoadditionalsinglebasedeletionsresultingin expressionof some functionalprotein. The presence of aplastic anaemia cannot be an absolute requirementfora clinicaldiagnosis of FA because of it relatively late onset in childhood in most cases. I n the absence, therefore,of congenital abnormalities as well as aplastic anaemia an increased sensitivity to DNA crosslinking agentscouldalsobeanindicationthatthepatientmighthaveNBS.Laboratoryfeaturesof NBSshouldbeinvestigatedincluding,definitively,theidentificationof NBS 1 mutations.I n addition,loss of fulllengthNbslproteinby Westernblotting,a substantiallyincreased sensi-tivity to ionizing radiation and the presence of translocations involving chromosomes 7 and14 in the peripheralbloodcan also beexamined. RelationshipofComplementatiotiGrouptoClinicalFeatures I t is unclear how much correlation exists between a FA complementationgroup and clinical findings.Differentinteracting factorswill affectany putativecorrelation.(1) Bearing inmind the factthatmanyof the FANC proteinscontributetoa single core complex, whichactivates FANCD2by monoubiquitination,it is possible that loss of any one of these proteins may have thesameeffectofdestabilisingthecomplex.Theinferenceofthismaybethattheclinical featuresassociated withloss of any one componentof thecomplex may be similar.O fcourse, itis possiblethatindividualcomponentsofthecomplexmayhavefunctionsinadditionto those of the core complex. Thismightlead to gene specificphenotypes.(2)FA-A patientsare thelargestgroupandmutationspecificeffects(e. g. , thepresenceof a particularmissense mutation allowing expression of some mutant FANC protein with some activity) may occur to give significantphenotypic variation withinthe group.(3)I tis quite likely thatgenetic back-groundor modifyinggenes will also contributetothe effectsof particularFA mutations. This may be more readily observed for the numerically larger complementationgroup A. (4) Finally, loss of FA proteins FANCDI (BRCA2), FANCD2 and the putative FANCJthat are not part of the FA protein core complex might be the ones expected to each give a consistent anddifferent phenotypetothe others. Comparison of PatientsinComplementationGroups FA-A, FA-C and FA'G (Table 4) Some comparison can be made between these three groups because they are commonenough toallowtheaccumulationofthenumbersofpatientsrequired.I twassuggestedthatFA-C, comparedwithFA-AandFA-Gpatients,hadasignificantlyearlieronsetofbonemarrow failure and poorer survival but there was no differencein the cumulative incidence of AML and MDS between FA complementationgroups A, Cand G."^There was also poorer overall survival for patients with at least one intron 4 (i^4A^CCIVS4+4A>Tmutation -a foundermutationin the Jewish population)^^'^^ or one FANCCexon14(R 548X or L554P)mutation. ^^ Molecular Mechanisms of FanconiAnemia Table 4.Features of patients in different complementation groups Upstream FA-A FA-B FA-C FA-E FA-F FA-G FA-I Downstream FA-J FA-D1(BRCA2) FA-D2 Frequency (%) 60-65 rare 10-15 rare rare - 1 0 rare rare rare rare SpecificClinicalFeatures Mutati onspecific poorersurvival NK Mutati onspecific poorersurvival morecongenitalabs CNSAbs. morecongenitalAbs earlieryBMfailure Carcinomas NK NK moresevere (tumoursearly) moresevere (congenitalabnormalities) Tumours AML carcinomas NK AML carcinomas ? NK AML NK NK AMl / r-ALLAA/ i l ms Medul l obl astoma NK References Kutleretal ,2003 Hermsen,2001 Kutleret al ,2003 Hermsen,2001 Faivreetal ,2000 Faivreetal ,2000 Kutleretal ,2003 Faivreetal ,2000 Levitusetal ,2004 Levitusetal ,2004 Offitetal ,2003 Hi r schet al ,2003 Wagneretal ,2003 Soulieret al ,2004 I n contrast, however, Faivre et al^reporting on EuropeanFA patients, suggested thatthere wasnodifferenceamongFA-A,FA-CandFA-Gpatientsforageofonsetofhaematological abnormalitiesalthoughthe cytopaenia was significantlyworseinFA-G . Faivre et alalso reportedthatFA-GhadahigherrateofAMLorMDSthanFA-AorFA-C.Therewasa reduced incidence of congenital abnormalities in FA-C patients, excluding those with the FANCC IVS4+4A>Tmutation,thaneitherFA-A orFA-G,thatwere similar.Morecongenitalabnor-malities were associated withthe FANCCWSA+4A>Ymutation. I n contrast, patients withthe /t^4A^CC322delGmutation had fewer congenital abnormalities. Patients with two nullFANCA mutations had an earlier age of onset of haematological abnormalities and shorter survivalafter diagnosiscomparedwithFA-ApatientswhoexpressedsomeFANCAprotein.Thebiallelic nullFA-A patientsalso showeda greaterproportionof AML/MDScomparedwithFA-A patientsexpressingsomemutatedFANCAprotein.TheFANCAproteinsassociatedwith differenttypesofFANCAmutationsshoweddifferentlevelsofcomplementationofMMC sensitivity,indicatingthatsome phenotypic variationwithinFA-A patientsmay be relatedto the type of mutationpresent. Patientsin the Rarer FA ComplementationGroups, FA-E and FA-F (Table 4) ThedifficultywiththerarergroupsFA-EandFA-Fistheabsenceofanysignificant numbersof patientstomakea real assessmentoftherelationshipbetweencomplementation group andclinicalfeatures.FaivreetaP^ suggestedthatcongenitalabnormalitiesmaybe morecommonin the FA-E andF patients. Interestingly,a significandyhigher proportion(3/ 5) of the FA-E patients presented withCNSmalformations. Clinical Features ofFanconiAnaemia Patients inComplementationGroups FA'D2 andFA-J(Table 4) Very few patients have a FANCD2mutation.Levitus et al^^ give a proportionof 8/241FA familiesintheD2complementationgroup.BecauseFANCD2isnota partoftheFAcore proteincomplex, but a target of it, patients with FANCD2mutationsmight be differentfrom the majority of FA patients. Soulier et al^ gave the clinical features of a group of fourFANCD2 patientsdiagnosedattheearly ages of 0. 3,3, 2. 8and6 years. I t was suggestedthattheyhad moreseverecongenitalabnormalitiesthanpatientsinothercomplementationgroups.The observation,by Faivre et al^^ of a higher rate of congenitalabnormalitiesin a combinedgroup of FA-D patientsis consistent withthis. O neFANCD2patientshowedevidence of FA rever-sion.^ Soulier et al^ also suggested that patients mutated for the putative FANCJgene,coding foranotherdownstreamprotein,mightalso have a more severe phenotype. AlthoughFA cells have beenexaminedforincreasedradiosensitivitythis is notacommon findingamongFA.^'^CellsfromFANCD2patientsdo,however,appeartoshowincreased radiosensitivity and have a defectin the ionising radiationinducible S phase checkpointas do A-Tcells. TheATMdependentphosphorylationofFANCD2onserine222isrequiredfor activation of the radiationinduced S phase checkpoint.This is additional to the FA complex dependent monoubiquitinationof FANCD2 on K561 as part of a second pathway in response todamage by DNAcrosslinkingagents. I nterestinglythemousemodeloi FancdZmictalso exhibitamoreseverephenotype.However,Fancd2'deficientmousecellsdonotshowan increasedcellularradiosensitivityorradioresistantDNAsynthesis.^Thisisdifferenttothe FANCD2cells butthe reason forthis differenceis notknown. Patients inComplementationGroup FA'Dl(Brca2)(Table 4) I thasrecentlybecomeclearthatFA patientswithbilallelicmutationofBRCA2havea severephenotype,atleastintermsofpredispositiontoearlyonsetofcancercomparedwith otherFA groups.^^'^^'^Two recent studies ona totalof11 patientshave highlightedboththe increasedriskof AML(6cases)andacute Tcellleukaemia(2cases)^^'^^andsolidtumours includingmedulloblastoma(2cases)and W ilmstumour(3cases)inthesepatients.^^ All11 patientsdevelopedatumourbeforetheageof5yearsandtwopatientshadtwotumours. Leukaemia occurred at a medianage of 2. 2 years compared witha medianage of onset of13. 4 years in other FA patients.'^^ I t is clear that differenttumourtypes can occur in the presence of the same BRCA2mutations; one of these patients hadboth W ilmstumourand T-ALL. W hat rangeoftumourswillbeassociatedwithbiallelicmutationofBRCA2remainstobeseen. Microcephaly,intra-uterinegrowthretardation,failuretothrive, shortstature andcafi aulait spotswerecommonfeaturestothesepatients.Severecongenitalabnormalitieswerelargely absentandonly4patientspresentedwithbonemarrowfailure.MutationsofBRCA2were demonstratedinallthepatientsandlossoffulllengthBRCA2proteininboththefamilies even thoughone familycarriedthe BRCA2 missense mutation7751T>C; L2510P.^^ Bearing in mind the consequence of loss of BRCA2 in the predisposition to some breast and other tumoursit might be anticipatedthat the familiesof these FANCDlpatients might be at some additionalrisk of tumours. A significantfamily history of breast cancer was noted in3/5 of thefamilies,althoughtherewas anapparentabsenceof anyincreaseinBRCA-2related tumoursinthe familiesstudied.^^ Anotherinteresting featureof cells fromthese patients was the striking increase,compared withotherFA patients,of spontaneouslyoccurringchromosomeabnormalitiesas well asthe high level of chromosome aberrations induced by DNA crosslinking agents. ^^ The presence of these spontaneoustypes of chromosome aberrationis reminiscentof those seen in otherBrca2 deficientcells.^'^ TheevidencesuggeststhatBrcaZmurinecellswiththedeletionofthe BRCA2-carboxyterminus are hypersensitiveto ionizing radiation(IR),^^althoughit is not clear whether this is true of the human BRCA"^' (CAPAN-1)cells. However, the increase in the radiosensitivitydoesnotappeartobe as greatas for A-Tcells. Interestingly,thereis nopub-lisheddataindicatingwhetherornotFANCD1cellsareunusuallysensitivetoI Ralthough, 10Molecular Mechanisms ofFanconi Anemia H irschetal^^ suggest,withoutdatabeingpresented,thatsomeoftheirFA-Dlcells showa marked sensitivity to ionising radiation. I t remains unclear, therefore, whether the FA-Dlcells are indeed unusually radiosensitive. I ndeed, it is not certain that this would be the case if these cells carry hypomorphicallelesofBRCA2. Theevidenceis thatBRCA2functionsinthesamecellularpathwayas otherFA proteins and, therefore,the cause of the more severe clinical appearance of BRCA2 mutantFA patients is intriguing? Evidencefor ModifyingMutations As expected,FA patients withina family,homozygousforthe same FANCmutation,may show similar clinical phenotypes.However, this is not always the case. Studying (1) the clinical variation between FA patients within other families(2) differencesbetween familieshomozygous for the same FANC mutation(3) the effectsof the presence of the same mutation on a different genetic background will all provide some evidence as to whether other factorsplay a role in the developmentof theFA phenotypes.I ntworelatedFA-A familieswithfouraffectedchildren, all sharingthesamehomozygousdeletiono FANCAexon43therewasmarkedvariationin theseverityofthecongenitalabnormalities^ betweenthetwofamiliessuggestingsome possibleeffectofgeneticbackground.Futakietalalsosuggestedthatmodifyinggenesor otherfactorscontributedtothe clinical phenotypesince they were able to show that Japanese patients with the FANCCIVS4+4A>Tmutationhad the same severity of clinical phenotype as other JapaneseFA patientswithdifferentTMA'^C mutations. Thiscontrastswiththemore severe clinical phenotypeassociated withthe i^A^CCI VS4+4A>Tmutationin Ashkenazi Jewishpatients. Conclusions FA patients present witha wide range of phenotypesandit is quite possible that otherrare complementationgroups remainto be defined.Fromthe data already available it seems likely that that some clear clinical differences will be established between those patients whose mutations affecttheFANCproteincorecomplexcomparedwiththosewhosemutationsaffectthe downstream proteins FANCDland FANCD2. A surprising findinghas been the predisposition toearlyonsettumoursofdifferenthistologicaltypethatoccursintheFANCDl(BRCA2) patients. Particularly intriguing are reports of Tcell tumours in these patients. T cell leukaemia alsooccurswithhighfrequencyinpatientswithA-Tandtheseobservationsmaybean indicationof a furtherinteraction between ATM and the FANC pathway, involvingFANCDl (BRCA2). Themajorchallengeis todefinethetargetsof theFANCproteins,tounderstand the FA pathway andreexaminetheclinical phenotypesin light of this knowledge. Acknowledgements TheauthorthanksCR -UK,TheKay KendallLeukaemiaFund,theLeukaemiaResearch Fundandthe Ataxia TelangiectasiaSociety forcontinuedsupport. References LTaylorAMR .Chromosomeinstabilitysyndromes.Bailliere'sBestPractice&R esearchClinical H aematology2001;14:631-644. 2.CleaverJE.DefectiverepairreplicationofDNAinX erodermapi gment osum.Nat ure1968; 218:652-6. 3.SasakiMS,Tonomur aA.AhighsusceptibilityofFancon' sanaemiatochromosomebreakageby DNAcrosslinkingagents.CancerR es1973;33: 1829-1836. 4.TaylorAM,H arndenDC,ArlettCFetal.Ataxiatelangiectasia:Ahumanmutationwithabnormal radiationsensitivity.Nat ure1975;258:427-9. 5.W eemaesCM,H ust i nxTW ,ScheresJMetal.Anewchromosomeinstabilitydisorder:The Nijmegenbreakagesyndrome.1981;70:557-564. 6.TaalmanR D,JaspersNG ,ScheresJMetal.H ypersensitivitytoionizingradiation,invitro,ina newchromosomalbreakagedisorder,theNijmegenBreakageSyndrome.Mut atRes1983;112:23-32. ClinicalFeaturesofFanconiAnaemia11 7.H ielJAPetal(I nternationalNijmegenBreakageStudyG roup).Nijmegenbreakagesyndrome. TheI nternationalNijmegenBreakageSyndromeStudyG roup.ArchDisChild2000;82:400-6. 8.H ernandezD,McConvilleCM,StaceyMetal.AfamilyshowingnoevidenceofH nkagebetween theataxiatelangiectasiageneandchromosomel l q22- 23.JMedG enet1993;30:135-40. 9.SavitskyK,Bar-ShiraA,G iladSetal.Asingleataxiatelangiectasiagenewi t haproductsimilarto PI -3kinase.Science1995;268: 1749-53. 10.StewartG S,MaserR S,StankovicTetal.TheDNAdouble-strandbreakrepairgeneh MR El lis mutatedinindividualswi t hanataxia-telangiectasia-likedisorder.Cell1999;99: 577-87. 11.CarneyJP,MaserR S,O livaresHetal.ThehMr el l / hR ad50proteincomplexandNijmegenbreak-agesyndrome:Linkageofdouble-strandbreakrepairtothecellularDNAdamageresponse.Cell 1998;93: 477-86. 12.VaronR ,VissingaC,PlatzerMetal.Nibrin,anovelDNAdouble-strandbreakrepairprotein,is mutatedinNijmegenbreakagesyndrome.Cell1998;93:467-76. 13.G arcia-H igueraI ,TaniguchiT,G anesanSetal.I nteractionoftheFanconianemiaproteinsand BR CAlinacommonpathway.MolCell2001;7:249-62. 14.NakanishiK,TaniguchiT,R anganathanVetal.I nteractionofFANCD2andNBSlintheDNA damageresponse.NatCellBiol2002;4:913-20. 15.D' AndreaAD,G rompeM.TheFanconianaemia/BR CApathway.NatR evCancer2003;3:23-34. 16.MeeteiAR ,deW interJP,MedhurstALetal.AnovelubiquitinligaseisdeficientinFanconi anemia.NatG enet2003;35:165-70. 17.W angX ,AndreassenPR ,D' AndreaAD.FunctionalinteractionofmonoubiquitinatedFANCD2 andBR CA2/ FANCD1inchromatin.MolCellBiol2004;24:5850-62. 18.LevitusM,R ooimansMA,SteltenpoolJetal.H eterogeneityinFanconianemia:Evidencefor2 newgeneticsubtypes.Blood2004;103:2498-503. 19.MeeteiAR ,LevitusM,X ueYetal.X -linkedinheritanceofFanconianemiacomplementation groupB.NatG enet2004;36:1219-24. 20.H owlettNG ,TaniguchiT,O lsonSetal.BiallelicinactivationofBR CA2inFanconianemia. Science2002;297:606-9. 21.DaviesAA,MassonJY ,McllwraithMJetal.R oleofBR CA2incontroloftheR AD51recombina-tionandDNArepairprotein.MolCell2001;7:273-82. 22.NakanishiK,Y angY G ,PierceAJetal.H umanFanconianemiamonoubiquitinationpathway promoteshomologousDNArepair.ProcNat lAcadSciUSA2005;102:1110-15 23.SchroederTM,TilgenD,KrugerJetal.FormalgeneticsofFanconi' sanemia.H umG enet1976; 32: 257-88. 24.SwiftM,Shol manL,PerryMetal.Mal i gnantneoplasmsinthefamiliesofpatientswi t h ataxia-telangiectasia.CancerR es1976;36: 209-15. 25.W oodsCG ,BundeySE,TaylorAM.Unusualfeaturesintheinheritanceofataxiatelangiectasia. H umG enet1990;84:555-62. 26.KutlerDI ,SinghB,SatagopanJetal.A20-yearperspectiveontheI nternationalFanconiAnemia R egistry(I FAR ).Blood2003;101:1249-56. 27.TischkowitzM,DokalI .Fanconianaemiaandleukaemia-clinicalandmolecularaspects.BrJ H aematol2004;126: 176-91. 28.W agnerJE,TolarJ,LevranOetal.G ermUnemutationsinBR CA2:SharedgeneticsusceptibiUty tobreastcancer,earlyonsetleukemia,andFanconianemia.Blood2004;103:3226-9. 29.LandmannE,Bluetters-SawatzkiR ,SchindlerDetal.Fanconianemiainaneonatewi t hpancy-topenia.JPediatr2004;145:125-7. 30.AlterBP.CancerinFanconianemia,1927-2001.Cancer2003;97:425-40. 31.R osenbergPS,G reeneMH ,AlterBP.Cancerincidenceinpersonswi t hFanconianemia.Blood 2003;101:822-6,(Erratumin:Blood.2003;101:2136). 32.AlterBP,G reeneMH ,VelazquezIetal.CancerinFanconianemia.Blood2003;101:2072. 33.ButturiniA,G aleR P,VerlanderPCetal.H ematologicabnormalitiesinFanconianemia:Anin-ternationalfanconianemiaregistrystudy.Blood1994;84:1650-5. 34.R osenbergPS,H uangY ,AlterBP.I ndividualizedrisksoffirstadverseeventsinpatientswi t h Fanconianemia.Blood2004;104:350-5. 35.TischkowitzMD,H odgsonSV.Fanconianaemia.JMedG enet2003;40:1-10. 36.R osenbergPS,SocieG ,AlterBPetal.R iskofheadandnecksquamouscellcanceranddeathin patientswi t hFanconianemiawhodidanddidnotreceivetransplants.Blood2005;105:67-73. 37.W ajnrajchMP,G ertnerJM,H umaZetal.Evaluationofgrowthandhormonalstatusinpatients referredtotheI nternationalFanconiAnemiaR egistry.Pediatrics2001;107:744-54. 38.AlterBP.Fanconi' sanaemiaanditsvariabiUty.BrJH aemat ol1993;85:9-14. 12MolecularMechanismsofFanconiAnemia 39.FaivreL,G uardiolaP,LewisCetal.Associationofcomplementationgroupandmutationtype withclinicaloutcomeinFanconianemia.EuropeanFanconiAnemiaR esearchG roup.Blood2000; 6:4064-70. 40.UnalS,O zbekN,KaraAetal.FiveFanconianemiapatientswi t hunusualorganpathologies.Am JH emat ol2004;77: 50-4. 41.H uberPAJ,Mat hewCG .FanconiAnaemia.I n:EelesR A,EastonDF,PonderBAJ,EngC,eds. G eneticPredispositiontoCancer.2nded.London:Arnold,2004: 180-192. 42.Duckworth-R ysieckiG ,H ul t enM,MannJetal.ClinicalandcytogeneticdiversityinFanconi' s anaemia.JMedG enet1984;21: 197-203. 43.G iampietroPF,VerlanderPC,DavisJGetal.DiagnosisofFanconianemiainpatientswi t hout congenitalmalformations:AninternationalFanconiAnemiaR egistryStudy.AmJMedG enet1997; 68: 58-61. 44.NewFiV,CaleCM,TischkowitzMetal.NijmegenBreakageSyndromediagnosedasFanconi anaemia.Pediatr.BloodCancer2005;44:1-6. 45.G enneryAR ,SlatterMA,BhattacharyaAetal.TheclinicalandbiologicaloverlapbetweenNijmegen BreakageSyndromeandFanconianemia.CUnI mmunol2004;113:214-9. 46.O ' DriscoUM,R uiz-PerezVL,W oodsCGetal.Asplicingmut at i onaffectingexpressionof ataxia-telangiectasiaandR ad3-relatedprotein(ATR )resultsinSeckelsyndrome.NatG enet2003; 33: 497-501. 47.ShimamuraA,deO caR M,SvensonJLetal.AnoveldiagnosticscreenfordefectsintheFanconi anemiapathway.Blood2002;100:4649-54. 48.SoulierJ,LeblancT,LargheroJetal.DetectionofsomaticmosaicismandclassificationofFanconi AnemiapatientsbyanalysisoftheFA/BR CApathway.Blood2005;105:1329-36. 49.W aisfiszQ,MorganNV,SavinoMetal.SpontaneousfunctionalcorrectionofhomozygousFanconi anaemiaallelesrevealsnovelmechanisticbasisforreversemosaicism.NatG enet1999;22: 379-83. 50.G illioAP,VerlanderPC,BatishSDetal.PhenotypicconsequencesofmutationsintheFanconi anemiaFACgene:AnI nternationalFanconiAnemiaR egistrystudy.Blood1997;90:105-10. 51.AdachiD,O daT,Y agasakiHetal.H eterogeneousactivationoftheFanconianemiapathwayby patient-derivedFANCAmut ant s.H umMolG enet2002;11:3125-34. 52.Duckworth-R ysieckiG ,TaylorAM.EffectsofionizingradiationoncellsfromFanconi' sanemia patients.CancerR es1985;45:416-20. 53.TaniguchiT,G arcia-H igueraI ,X uBetal.ConvergenceoftheFanconianemiaandataxiatelang-iectasiasignallingpathways.Cell2002;109:459-72. 54.H ought al i ngS,Ti mmersC,NollMetal.EpithelialcancerinFanconianemiacomplementation groupD2(Fancd2)knockoutmice.G enesDev2003;17:2021-35. 55.H irschB,ShimamuraA,MoreauLetal.AssociationofbialleUcBR CA2/ FANCD1mutationswith spontaneouschromosomalinstabilityandsolidtumorsofchildhood.Blood2004;103:2554-9. 56.O ffitK,LevranO ,MullaneyBetal.Sharedgeneticsusceptibilitytobreastcancer,braintumors, andFanconianemia.JNat lCancerI nst2003;95: 1548-51. 57.VenkitaramanAR .TracingthenetworkconnectingBR CAandFanconianaemiaproteins.NatR ev Cancer2004;A:2GG-7G. 58.MorimatsuM,DonohoG ,H astyP.CellsdeletedforBrca2CO O Hterminusexhibithypersensi-tivitytogamma-radiationandprematuresenescence.CancerR es1998;58:3441-7. 59.PatelKJ,Y uVP,LeeHetal.I nvolvementofBrca2inDNArepair.MolCell1998;1:347-57. 60.Y uanSS,LeeSY,ChenGetal.BR CA2isrequiredforionizingradiation-inducedassemblyof R ad51complexinvivo.CancerR es1999;59: 3547-51. 61.TamaryH ,DganyO ,ToledanoHetal.MolecularcharacterizationofthreenovelFanconianemia mutationsinI sraeliArabs.EurJH aematol2004;72:330-5. 62.KocA,PronkJC,AlikasifogluMetal.Variablepathogenicityofexon43del(FAA)infourFanconi anaemiapatientswithinaconsanguineousfamily.BrJH aematol1999;104:127-30. 63.FutakiM,Y amashitaT,Y agasakiHetal.TheI VS4+4AtoTmutationofthefanconianemia geneFANCCisnotassociatedwithaseverephenotypeinJapanesepatients.Blood2000;95:1493-8. GA.KutlerDI ,AuerbachAD.FanconianemiainAshkenaziJews.FamCancer2004;3:241-8. 65.H ermsenMA,X ieY,R ooimansMAetal.Cytogeneticcharacteristicsoforalsquamouscellcarci-nomasinFanconianemia.FamCancer2001;1:39-43. GG.W angX ,D' AndreaAD.TheinterplayofFanconianemiaproteinsintheDNAdamageresponse. DNAR epair(Amst)2004;3:1063-9. CH APTER2 TheGeneticBasis of Fanconi Anemia Grover C. Bagby, Jr.* Introduction Seventy-fiveyearsago,Dr.G uidoFanconireportedthreesiblingswhoexhibitedboth congenital defects and aplastic anemia.Since then, we have learned that Fanconi anemia (FA)isararemultigenicdisorder(aprevalenceof1-5permillion"^),thatpredisposes childrenandadultstolife-threateningbonemarrowfailure,myelodysplasia,^acute nonlymphocyticleukemia(AML),andcertainepithelialmalignancies.'^So far,the sine qua non of this diseaseis cytogeneticinstabilityin vitroafterexposureof FA cells tobiftinctional alkylatingagents.'I ndeed,thecurrentdiagnostictestforFanconianemia,quantificationof chromosomalbreakage responses to alkylating agents, is based onthis feature(reviewedinref. 10).Classic clinical featuressuch as growthretardation,small head size, cafi-au-lait spotSyand radialray defectscanbe strongdiagnosticclues,butFA canoccurinpatients withoutcon-genital defectsandcan be clinically ascertainedin adulthood.I nfact,some patients with very minimalbloodcountabnormalitieshavebeenidentifiedonlybecausetheyweresiblingsof knownFA patientsand were tested forthatreason. Consequendy,there is substantialpheno-typicheterogeneityinFAandwhilesomeofitcanbeexplainedbygeneticheterogeneity, certainof theclinical consequencesare the result of gene-environmentinteractions. GeneticHeterogeneity Somatic cell fusionstudies have defined11 complementationgroups, FA-A, B, C, Dl ,D2, E,F, G ,L,Iand J.Allelevencanbeaccountedforbymutationsofa geneuniquetothat groupand nine of the genes have been cloned^^'^^(Table1). Some of the proteins encoded by thenormalFANGgenes containdomainsthatsuggest some functions(FANCGforexample has a number of tetratricopeptide[TPR] repeats). However, with the exception of FANCDl, nowknowntobeidenticaltoBRGA2,^^ thegenesandproteinstheyencodehavenostrong homologiestoone anotheror tootherknownproteins. TworecurringthemeshaveemergedfrompublishedstudiesonthefunctionoftheFA proteins;(1)thatthey functiontoprotectagainstgenotoxicstress atleastinpartbyforming complexes witheach other and facilitatingmonoubiquitinationof FANG D2, and(2) thatthe proteins also interact functionallywith other proteins governing survival-signaling pathways. ^^ W hile this second functionof FA proteinsinvolves formationof complexes, they are probably complexes composed of molecules not foundin the so called "core FA complex" that consists of FANGA, FANG B, FANG G , FANG E, FANGF, FANG Gand FANGL. I n fact,as will be men-tionedin more detail below, the definitionof the "core complex" is undergoing somesubstan-tial revision. *Grover C. Bagby, Jr.OHSUCancerInstitute, Departments of Medicine and Molecular and MedicalGenetics, OregonHealth and Science University,Portland, Oregon 97239, U.S.A. Email: grover@ohsu.edu Molecular Mechanisms of Fanconi Anemia,editedby ShamimI. Ahmadand Sandra H .Kirk. 2006Eurekah. comandSpringerScience+BusinessMedia. 14Molecular Mechanisms of FanconiAnemia Table1. Gene FANCA FANCB FANCC The Fanconi anemia genes FANCD1(BRCA2*) FANCD2 FANCE FANCF FANCG FANCL(PHF9) FANCI FANCJ Although mutations Prevalence of Mutations in Patients with FA (%) 70 1 10 1 1 5 2 10 1 Notknown Notknown Chromosomal Location 16q24.3 Xp22.31 9q22.3 13q12.3 3p25.3 6p21.3 11 pi 5 9p13 2p16.1 Notknown Notknown Exon Number 43 10 14 27 44 10 1 14 11 Notknown Notknown aBRCA2nullgenotypeis an embryoniclethalphenotype,certain that lead to C-terminaltruncations, Amino Acid Residues (kDa) 1455(163) 859(95) 558 (63) 3418(384) 1451(155,162) 536 (60) 374 (40) 622 (48) 373 (43) Notknown Notknown homozygousBRCA2 lead toFA of the D1 complementation group. The iMM7 Genes Reviewed below are selected features of each FANCgeneand the proteins they encode. The chromosomallocationandexonnumberof the genes andaminoacidresidues andmolecular massoftheencodedproteinsarelistedinTable1. AFanconiAnemiaMutationDatabase, containingmanyFAmutations(butnotall),isavailable(www.rockefeller.edu/fanconi/mu-tate). Currendy, ascertainmentof novel mutations is most oftendone by DH PLCanalysis and sequencing of genomic DNA and cDNA. Apart fromfamily studies, to confirmthat an abnor-malityrepresentsamutation,complementationanalysis(inwhichthewildtypecDNAex-pressedinmutantcellscorrectsMMC/DEBsensitivitybutthemutantcDNAdoesnot)is mostconvincing.However,inlightoftheinformationnowextantonkeyregionsofFANC genes,somesequencevariations(especiallylargedeletions)canbeassignedasmutationsby deduction. FANCA Mutationsofthis geneareby farthemostcommoninpatientswithFA, accountingfor 65%of all cases. The spectrumand scope of discrete mutationsof this gene are immenseand include nonsense, missense, and splicing mutations as well as microdeletions,microinsertions, andduplications. ^^ Theremay be morethan250differentmutantFANCAalleles, manyof whicharelargeintragenicdeletions,^^andonlyafewarecommonespecially c. lll5_1118delTTG G(2%of FANCAalleles)andc. 3788_3790delTCT(5% ofFANCA alleles).^^ Theproteinparticipatesas a memberofthecore complexrequiredforFANCD2 ubiquitination,"^^andbindstoBRCAl,'^^ butthefunctionalrole it plays inthesecomplexes areunknown. Posttranslationalmodificationsof FANCAoccurinresponsetoenvironmentalcues. For example, FANCA is redox-responsive and multimerizes withFANCGin response to oxidative stress."^^ PhosphorylationofFANCAalsooccursinnormalcellsbutnotinmostFA cells. I n vitro phosphorylationstudies identifieda cytoplasmic serine kinase embedded in the core com-plex (sensitive to wortmannin)that phosphorylatedFANCA on serines.The functionalcon-sequences of this post-translational change and whether this putative FANCA kinase is the one thatseems tobe modulatedby AKTareunknown. The Genetic Basis ofFanconi Anemia15 Like FANCC, FANCA also seems to participate directly in support of survival signal trans-ductionpathways. For example, FANCA interacts withI KK2 and may thereby integrate with theIkappaB kinase(IKK)signalsome in a functionalway. Interestingly,this IKK2interaction isreportedlyrequiredforstimulus-dependentphosphorylationofotherproteinsintheFA complex.FANCAalso interacts withBR G l,a subunitof theSW I /SNFcomplex,andmay regulatestress-inducedchromatinremodeling."^"^I ntriguingly,inFANCAmutantcellsthe molecularchaperoneG R P94isfoundinaBRG-1associatedcomplexbutisnotfoundin normal or complementedcells."^^ The significanceof the phenomenonis unexplainedbut it is one of three specificexamples in whichaberrantor nonnativeproteincomplexes are foundin FA cells.I nsomeinstances(e.g.,inthecaseofFANCC)thereareaberrantsignalingconse-quencesthatattendthe formationof these complexes. GiventhatFANCA and FANCCboth also associate withmolecularchaperones,it is quite possible thatthese andotherFA pro-teinsare primarilycochaperonesthatmaintaintheproperstructuralconformation(andstate ofactivationorinactivation)of proteinsthatprotectcellsfromenvironmentalstresses. This theoreticalmodelwouldfitwitheverybindingphenomenonandbiologicalfunctionassay described to date forthe FA proteins. For example, the core complex could create a scaffoldfor the cross-linking agent-inducedactivationof an ubiquitinligase (possibly FANCL).^^ FANCB O ne of the most recendy discovered FA genes was identifiedusing a proteomics approach. ^^ FANCB was discovered by mass spectrometric analysis of a 95 kDa protein found in a multimeric FAcomplex.I thas10exons,7ofwhicharecodingexons(3-10). TheCterminusofthe proteincontainsaputativebipartitenuclearlocalizationsignalandispresentinnuclearex-tracts of normal cells butin mutantFA-A cells, FANCBis found,like FANCL, in the cytosol. That FANCB is required for FANCD2 monoubiquitination^^confirmsthat FANCB is a func-tionalmemberof thecore complex. The gene encoding this protein is located at X p22. 31 and subjectto X -chromosomeinacti-vation.^I nthecasesdescribedtodatetherearedifferentmutationsinthisgeneincluding insertionalframeshiftsinexon3and8,a deletionalframeshiftinexon8,anda large5'and promoterdeletion.I t has beenproposedthatas a single active copy gene, theonly X-linked FA gene, it may represent a vulnerability point forthe genome of cells fromobligate heterozy-gotes. An alternative view is that this proteintoo will prove to be multifunctionalandrequired forcellsurvival.I ndeed,studiesofthemethylationstatusofthegeneshowedthatFANCB inactivationseems to be skewed towardthemutated allele.^^ I t is increasingly evident that the functionofFanconiproteins may be lost during multi-step carcinogenesisbutitisnotclearwhetherthelossoccursearlyorlateincarcinogenesis. Basedonserialstudiesof cytokineresponsesofcommittedhematopoieticprogenitorcellsin humanswithFA,wehaveproposedthatacquiredlossofFAgenefunctioncannotbean initiating event (such a cell would lose a competitive advantage) and that other somatic changes mustoccurfirsttoprotectsuchcells fromtheapoptoticconsequencesof FA loss.^^'^^'^Two recent findings have supported this model indirecdy. First, apoptotic responses are indeed blunted in marrow cells of FA knockout mice with clonal evolution.^'^^ Second, the observation that in FANCB heterozygotes, X inactivation is skewed in favor of the mutated allele, indicates that the mutantcells are less fit to compete withthe nonmutantones during development. A FANCB knockoutmouse will be requiredto determine at which stage of developmentinheterozygotes the mutantFANCB cells are lost. There are likely othernonDNAdamage related functionsof theFANCBgenegiventhattheaffectedmaleshaveclassicFAphenotypesincludingbone marrow failure,butno data have been developed yet in supportof thisexpectation. FANCC This was the first of the FA genes tobe cloned. ^^ Tentofifteenpercentof FA cases canbe accountedforbymutationsofthisgene.Althoughthereareanumberofheterogeneous 16 Molecular Mechanisms of FanconiAnemia Table2.SomenonFAFA-bindingproteins FAProteinNonFABindingPartnerFunctionalRelevance BRCA2 FANCC FANCC RadSl ' STAT1 hsp70 FANCC FANCC FANCC FANCC FANCC FANCA FANCA FANCA FANCC FANCD2 NADPHcytochrome p450reductase^ ^FAZF^31,132 GRP94^^3 cdc249 GSTP1^^ BRG127 Al phaspectrinand XPF^^^ SNX5^^^ CYP2E^^7 USP1 ExperimentsdemonstratingthatFANCD1mutantcells exhi bi tedaberrantdamage-inducedRadSInuclear focusformati on[79]ledtoidentificationofBRCA2as a FA-D1compl ementi nggene.MutationsofBRCA2 thatleadtoFA disrupttheRad51bi ndi ngregion.^FANCCfacilitatesacti vati onofSTATsbycytokines^"^ andprobablypromotessurvivalofhematopoieticcells bydoi ngso. FANCC/hsp70bi ndi ngsuppressesPKR activation^^'^^ andtherebypromotessurvivalofhematopoieticand embryonalcells. Unknown. Unknown.Possiblechromati nremodel i ngfuncti on. TherelevanceofFAZFis suggestedbyitsconsistent expressioninpri mi ti vehematopoieticcells.^^^ Unknown.GRP94hasanti-apoptoticacti vi ty(93)and FANCCmayfacilitatethatfuncti on. UnknownbutresultsofKruytetaP^"^ suggestthe interactionmaybefuncti onal Unknown Unknown.Possiblechromati nremodel i ngfuncti on (BRG1is a subunitofthechromati n-remodel i ngSWI/ SNFcompl ex). Unknown. Unknown.Possibleco-chaperoneeffectofFANCAin SNX5-dependentreceptortraffickingbetween organelles.^^^ Unknown USP1bindstoanddeubiquitinatesFANCD2and probabl ymodulatestheFApathwayinthisway.^^ mutationsof this gene, two mutations account formost of the known FANCC mutations; the firstinexon1 (322delG )andtheotherinexon4(c. 711+ 4A>T,alsountilrecendytermed "I VS4+4A-T" is a single base change in the fourthintronic base that results in deletion of exon 4). The322delGmutationis hypomorphicforsome FANCC ftmctions,^^ likely because of a downstreamreinitiationsitethatgivesrisetoatruncatedprotein.Thec. 711+4A>Tis unique to patients of Ashkenazi Jewish ancestry in which populationthe carrier frequencyis 1/ 100.^2'^^ Because it was the first gene cloned more is known about the functionof FANCC thanany otherFA gene.Manyofthelessonslearnedaboutthisproteinwilllikelyholdgenerallyfor mostoftheothermembersofthecorecomplexaswell.FANCCisamemberofthecore complex,^^'"^^ and bindsto FANCE which facilitatesnucleartranslocationof FANCC.^'^'^^ At least one nuclear functionof this proteinis to facilitatethe FANCD2/BR CA1interactionin S phaseandincellsexposedtocross-linkingagents. Anothermightbetoinfluencecellcycle controlbecausetheFACprotein(butnotamutantprotein)coimmunoprecipitateswith cyclin-dependentkinase, cdc2.O therFANCC-interactingproteinsare listedin Table 2. The Genetic Basis of Fanconi Anemia17 FANCCIsRequiredforS urvivalofHematopoieticCells I n mice, the most consistent FA phenotype is hypogonadism,^^'^'^ but in humans the single dominantearly life-threateningfeatureofFA phenotypeis hematopoieticfailurethatresults fromexcessiveapoptosisinhematopoieticcellpopulationsincludingmyeloidanderythroid progenitors. ^^'"^^'^^TheFANCCproteinsets a highapoptotic thresholdin normalcells and apoptotic responses of FA cells exposed to certain extracellular apoptotic cues (interferongamma, tumornecrosisfactoralpha,mi p-l a,faslieand,anddsRNA)areexaggeratedin vitroandin vi vo10'28.29,56,58,60,63-65^^^^ Forthenuclearcore FA complextoaccountforthehematopoiesis-specificanti-apoptotic functionof FA proteins,itmightserve to directly governthe expressionof genesthatencode intra-orextracellularsurvivalfactorsspecificallyforhematopoieticcells. Analternative view, onebettersupportedbyexperimentalevidence,isthattheFANCproteinshavefunctions independentoftheircapacitytoform"thenuclearcorecomplex"andthatsomeoftheFA proteinsmighteven work entirely ontheirown(reviewedinref10). FANCCandSTATS ignaUng We determinedmore than a decade ago that FANCC is required for survival of hematopoi-etic cells.^^ O urresults predictedthatthe FANCCproteineither facilitatedsurvival signals or suppressed apoptotic cues. I t turns out that FANCC plays bothroles. For example, FANCC is requiredforoptimalactivationofSTATmoleculesandthe Jakkinasefamilymember Tyk2, therebyfacilitatingcell survivalandpreservinginterferonresponses, ^^'Recentlyithasbeen foundthatthedisruptionofthissignalingpathwayunderliesa subtle T-celldefectinFancc knockoutmice.The hematopoietic support functionof CD4cells is also perturbed(Fagerlie S, manuscriptin preparation)emphasizing the multi-factorialnature of marrow failurein FA. Thatis, notonly are myeloidanderythroidprogenitorcells hypo-responsivetosurvival cues, lymphoidcells that supportsurvival of progenitorsare defectiveas well. FANCC,hsp70,andPKR FANCCalso governs the apoptotic thresholdby suppressing pro-apoptotic signals. I t sup-presses the state of activationof the double strandedR NA dependentprotein kinase, PKR,by binding to hsp70 (like a cochaperone) and facilitating the association of PBCR and hspyO.^^'"^^' PKRexistsinaninactivemonomericgroundstateinnormalcellsbutisconstitutivelyacti-vated in FA-C cells.As a result of this aberrant state of activation of PKR, exposure of mutant cells to I FN-gamma andTNF-alphahyper-activates PKR and accounts for earlier observations thatFA-CprogenitorcellswerehypersensitivetoI FN-gammaandTNF-alpha. ^^I ndeed, PKRactivityisnowknowntobeincreasedinprimarybonemarrowcellsofpatientswith FA-C, FA-A, and FA-G.^^'^^ Interestingly,mutations of FANCC, FANCA and FANCGresult inenhancedFANCC/PKRbinding,andwhileitis unclearthatthisaberrantbindingperse results in PKR activation(possibly by facilitatinga structuralchange residting in groundstate PKR dimerization)the results do suggest that nonnative FA complexes can create serious patho-physiologicalconsequences. FANCCandO xidativeS tress I thasbecomeincreasinglyclearthattheFAproteinsplayaroleincontrollingcellular responsestoavarietyofextracellularchallengesandstress;biotic,oxidative,andchemical. Resultsofanongoingmulti-institutionalFA transcriptomeconsortiumsupportthisnotion. Results of the study will be broadly shared withthe scientificcommunityin the future.There isalsogoodbiochemicalevidencethatFA proteinsinfluencethestatesofactivationofkey mediatorsof cellular stress responses, including G STPlandASKl. Manyinvestigatorshavelongheldthata fundamentaldefectofFA cellsis intoleranceto oxidative stress.'I n support of this notion, Buchwalds group recently reported thatFANCC enhancesthefunctionofG STPlincellsexposedtoinducersofapoptosis.^G STPlisan 18Molecular Mechanisms ofFanconiAnemia enzymethatdetoxifiesby-productsof redoxstressandxenobiotics. W hileFANCCdoesnot direcdy interact with G STPl,its influence on this molecule may play a central role in tolerance ofextracellularcuesofmanykinds.H anelineandhercolleaguesrecendyobservedASKl hyper-activationin H2O2 -treated Fancccells and used gain- and loss-of-functionanalyses to confirmthat the hypersensitivity of Fancc mutant cells to oxidative stress was mediated, at least in part,throughalteredredoxregulationand ASKlhyperactivation/^ FANCCIsMultifunctional StudiesonSTATsignalingdefectsinFAcellshavealsopermittedthedevelopmentof structure-ftmctionstudiesthatprovethatFANCCismultifiinctional.Specifically,acentral, highlyconserveddomainofFANCCis requiredforfiinctionalinteractionwithSTATland conservative mutations of this domaininterfere with STAT signaling functionsof FANCCbut thesemutationshavenoimpactonthecapacityofthedomainmutanttocomplementin MMCassays.^ Anothermutant(322delG)wasunabletocomplementinMMCassaysbut didcorrecttheSTAT signaling defect.Interestingly,thismutationoccursinFA patientswho have mild disease andmildhematopoieticdefects. S tudiesonFANCCand theMolecularB asisofMosaicism Some patients with FA exhibit mosaicism in hematopoietic cells. These cases are ascertained by observing 2 subpopulationsof lymphocytes, one sensitive and one resistant tocross-linking agents. Studies on FANCC structure have revealed mechanisms by which mosaicism occurs in suchpatientsandcanbeusedasamodelformosaicisminothercomplementationgroups. Mosaicismcanderivefromrecombination"^^orcompensatorysequencealterationsincis.^^ Twopatternsofrecombinationweredescribedinhaplotypeanalyses;thefirstwas a single intragenic crossover between the maternally and paternally inherited mutations and the second was likely geneconversion.I nsupportof thehematopoieticsurvivalfiinctionof FANCC,in the majority of the mosaic patients studied, the blood counts were only minimally suppressed.^ I nsummary,FANCCisamultifiinctionalproteinthatprotectscellsfromthegenotoxic consequencesofcross-linkingagentexposurebycomplexingwithothermembersoftheFA corecomplextoparticipateinthe"linearpathway".^^Italso supportssurvivalof cells inthe ground state and cells exposed to a variety of biological cues that induce apoptotic responses. It does this by participating in at least three signaling pathways (STAT, PKR, and ASK signaling) at least in partbyfiinctioningvery muchinthe same way a cochaperone wouldfiinction.We believe thatin time these nongenotoxicityfunctionsof FANCC will be matchedby likefunc-tions of the other FA proteins, some of which have domains(e.g., the TPRmotifsinFANCG ) thattheoreticallymeet standardsforcochaperonefunctionality. FANCDl(BRCA2) Two observations converged to reveal the identity of the FANCDlgene. First, it was known thatBRCA2bindsRad51,^^'^^andsecondthatFANCDlmutantcellsexhibitedaberrant damage-inducedR ad51nuclear focusformation.^^These observations led to identificationof BRCA2asaFA-Dlcomplementinggene^"^ andtheconfirmationthatFA allelesofBRCA2 disruptthe R ad51binding region. ^^ This is nota commonallele and because truly nidimuta-tionsof BRCA2are lethalina murinemodel,^^themutationsof BRCA2thatleadtoFA are likely hypomorphic. The few FA-Dlpatients identifiedto date have presented with early-onset malignanciesof bothhematopoieticandnonhematologic types.^"^ MonoubiquitinatedFANCD2seems to promote loading of BRCA2 into multimeric com-plexes on chromatinby binding to the Cterminus ofBRCA2. ^^Twoseparate sites onBRCA2 also interact with FANCG . ^Using the I-Scel endonuclease to introduce a double-strand break at a specificchromosomallocus, Jasins group foundthatBRCA2 mutantcell lines are recom-binationdeficient.^^Therefore,itislikelythatthesecomplexesarerequiredfornormal homology-directedDNArepair. The Genetic Basis ofFanconi Anemia19 FANCD2 Thegeneclonedby MarkusG rompe s groupatO regonH ealthandScienceUniversity^ led tothe developmentof the"linearpathwaymodel" forintegrationof FA proteinfixnction. FANCD2associates withR AD51andBR CAlduringS phase^^ butgiventhesheersizeand likely complexity of these multimeric complexes, the associationof these proteins per se is not sufficientto facilitate DNA repair by homologous recombination. Ubiquitinationof FANCD2 requires the core complex but efficientubiquitinationalso requires the ATR checkpointkinase andR PAl.^ Recentlythedeubiquitinatingenzyme,USPlwasfoundtobindtoFANCD2 (Table 2)andsuppress expressionof the ubiquitinatedform. Thereis a gooddeal of uncertaintyaboutthefunctionof themonoubiquitinatedformof FANCD2andits functionalrelationshipwithBR CAlandBRCA2. Forexample,there were conflictingreports on the influenceof the FA pathway on R ad51 and BRCA2 focusformation incellsexposedtoionizingradiation.O hashietal^^ recentlyutilizedBRCA2andFANCD2 deficient cells and cells treated with siRNAs specifically targeting BRCA2, R ad51, and FANCD2 and developed data suggesting that FANCD2 does not have a direct role in BRCA2 andR ad51 associated homologousrecombinationrepairafterDNAdamage. Thisquestionstillrequiresfurtherevaluationbecause(1)survivalassays wereutilized(2) FA cells are hypersensitiveto interferon(3)of the knowncapacity of some siRNAstoactivate interferonresponses,^^andthis pitfallwas notexperimentallyruledout. FANCD2andDiagnosisofFanconiAnemia I nactivatingmutationsinallFAgenesbutBRCA2leadtofailureofaccumulationof FANCD2in damage-inducednuclear foci'^^and FANC alleles of BRCA2 interdictaccumula-tionof R ad51 in such foci.^^ Therefore,althoughit is currendy impractical, screening cells for FA lesions could involve microscopic assessments of damage-induced nuclear foci forFANCD2 and R ad51. FANCD2 immunoblots that distinguish the ubiquitinylated and nonubiquitinylated formsof FANCD2mightalso be usedtoimplicateinactivatingmutationsof any knownFA geneexceptBRCA2.^2I nthecontextof anunambiguousFA phenotype(by MMCassays in theappropriateclinicalsetting),ifFANCD2isubiquitinylatedthelikelycomplementation groupswouldbeFA-DlorFA-J.BRCA2mutantscouldbethenidentifiedusingBRCA2 immunoblotswhich shouldreveal only truncatedversions of theprotein. FANCE A rare FA gene, FANCE maps to 6p22-p21^^ and encodes a 536 amino acid protein that is partofthecorecomplex^'butplaysaroleintransportingcytoplasmicFANCCintothe nucleus.^ Mutationsof FANCEinclude a 355C-Ttransitionin the FANCEgene, leading to a glnl 19-to-ter(Q119X )nonsense change, a G-to-A change at position-8 in intron5, result-inginfalsesplicingandinsertionof 6nucleotidesfromintron5, includinganin-framestop codon,and a 421C-Ttransitionresulting inan argl4l-to-ter(R141X)nonsense change.^^ FANCF Discoveredbycomplementationcloning,de W interetal foundthattheFANCFgeneis intronless and encoded a peptide with homology to the Nterminus(R NA binding domain)of theprokaryotic R NA-bindingproteinR O M.FANCFis largely nuclearandis a memberof thecore complex. Mutationsof FANCFdescribedto date are mosdy deletions including a 23 base-pair deletion of nucleotides 230-252, a 47-bp deletion (349-395), a 2 bp deletion (484-485), O thermutations include a 16C-T transition resulting in a gln6-to-ter nonsense mutation,and a 327C-Gtransversion(yrl09-to-ternonsensemutation). Theproteinencodedby this gene may functionas a super-scaffold,organizingtheforma-tionof multimeric FA complexes. TheC-terminusof FANCFinteracts directly withFANCG and may facilitate the assembly of other FA proteins into the core complex (or othercomplexes for that matter).^^ The N-terminus of FANCF seems to stabilize the FANCA/FANCGinterac-tionandis also requiredforbinding of the FANCC/FANCEcomplex.^^ 20Molecular Mechanisms ofFanconi Anemia FANCG By functionalcomplementationof the Chinese H amster ovarian cell line UV40, Liu et al cloned X R CC9a gene thatconferredresistance to bothhygromycinandmitomycinC.Later FANCGwas shownto be identical to XRCC9^'^ whichhad been localized to9pl 3. Theputa-tive 622-amino acid nuclear and cytoplasmic protein is a member of the core complex'^and is phosphorylatedat serines 7, 385and387. Survival signaling functionsof FANCGinvolve suppression (in collaboration withFANCC and FANCA)of the proapoptotic double strandedR NA-dependentprotein kinase PKR.^^I n FANCGmutantcells, binding of FANCC and PKR is increased.^I n keeping withthe theme thatnonnativeinteractionsof FA proteins withsignaling proteinsin FA mutantcells contrib-utetopathogenesis,PKRactivityis increasedinbonemarrowcells of patientswithFanconi anemia withmutationsinthe FANCC,FANCA,and FANCGgenes. There are numerous FANCGmutations. Most result in proteintruncationsbut the sites of mutationare notclustered.In9 G ermanFAG patientsthere was a 313G -Ttransversionin 8of18(44%)mutatedalleles.^^^I n7Portuguese-BrazilianprobandsAuerbachetal^^^ re-portedIVS8AS-2A-G.ThesameteamalsoreportedI VSllDS+lG -Cin7French-Acadian probands,1794-1803delin 7 Europeanprobands, andI VS3+1G >C(five Koreanor Japanese probands)andsuggestthatthePortuguese-Brazilian,French-Acadian,andKorean/Japanese mutations were likely to have been present in a foundingmember of each of these populations. I n black populations of sub-Saharan Africa,the incidence of FA is > 1/40,000 and 82% of cases carry die same FANCGmutation(c. 637_643del TACCGCC). ^^^ FANCGproteinis a memberofthecorecomplex,associateswithFANCAandFANCF, and dimerizes with FANCA in cells exposed to oxidative stress.FANCGhas 6 or 7(depend-ingonthestringencyoftheconsensussequencedefinition)tetratricopeptiderepeats,ele-ments that facilitateprotein-proteininteractionsand,interestingly, are domainsused by other cochaperones to bind to hsc70^^^ and hsp90,'^^'^^'^^^ raising the distinct possibility that FANCG , like FANCCmay integrate withheat shockresponses as a cochaperone. FANCH Thereis noFANCHgene. It was predictedfromcomplementationgroupanalysiscarried outoncells later shownto be FANCA mutants. ^^^ FANCI and FANCJ Neitherofthesetworecentlyidentifiedgeneshasbeenclonedtodate. Thattheyexist is suggestedbysomaticcellfusion/complementationanalysesand/orgeneticdata.Using immunoblotanalysesforFAcorecomplexformationandFANCD2mono-ubiquitination, Levitus et al demonstratedthat bothFA-I and FA-J cell lines formeda core complex, that FA-I cellswerenotcapableofubiquitinatingFANCD2,andthatFA-Jcellsdidubiquitinate FANCD2. ^^ ThisplacedthetheoreticalproteinencodedbythemutatedgeneinFA-Jcells "downstream" of the FANCD2ubiquitinationstep. FANCL This was the first of two FA genes(FANCBbeing the other)identifiedusing a proteomics approach.Specifically,Meeteietal^^ identified,usingmass spectrometry,a 43kDalFanconi complexassociatedproteinasPH Dfingerprotein-9(PH F9).The373-aminoacidprotein contains 3 W D40repeats and a PH D-type zinc finger motif and is clearly a key component of the FA core complex. The protein has ubiquitinligase activity and may play animportantrole inthemonoubiquitylationof FANCD2. ^^I neffect,theothermembersof the corecomplex may serve as a scaffoldthatpermits thepresentationof FANCLto FANCD2and possibly to other proteinsas well. I n fact,taking intoaccountthe degree to whichFANCD2is conserved throughoutevolution^it seems not sufflciendyparsimoniousthat a complex of 7 FA proteins hasevolvedsimplytofacilitateoneposttranslationalchangeofFANCD2.Experimentsare The Genetic Basis ofFanconi Anemia21 now underway in our laboratory to identify other substrates that might be ubiquitinatedby the FA complex. The mutation described by Meetei et al^^ was a deletion of exon11 that removed the part of the W D40repeatandtheentireconservedPH Dfinger.Itarose by homozygousinsertionof 177bpattheintron10/exon11 splice junction. FA ProteinComplexes The FA ^'Nuclear Core Complex*' W iththe exception of FANCD2and BRCA2, most of the FA proteins are thought toform a large multi-protein complexes.^^'^ ^^'^ ^^ I nactivating mutations of FANCA, FANCB, FANCC, FANCEandFANCGproteinsreduceassembly,stability,and/ornucleartranslocationofthe multi-subunitFA protein"core" complex.'^^ O nefijnctionof the core complex has been deduced by nicely-designedstudies onisogenic FA mutantcelllines. ThesestudieshaveclarifiedanemergingrelationshipbetweenFANG proteinsandsome functionsof BR CAl,BRCA2andR ad51.First,as mentionedabove,cer-tainBRCA2(FANCDl)mutationscanresult inthe FA phenotype(FA-Dl)^'' andthemuta-tions result infailureof R ad51 to localize in damage-inducednuclear foci.Second,it seems clearthatinactivationofanyoneofthecorecomplexproteinsinterdictsformationofthe completeFAcomplex,oneconsequenceofwhichisreducedubiquitinylationand BR CAl-colocalizing capacity of the nuclear FA proteinFANCD2.'^Therefore,accumulation of BR CAland R ad51 in damage-inducednuclear foci is required to protect cells fromdamage induced by cross-linking agents. Moreover, to accumulate in these foci, BR CAlrequires FANCA, B, C,G,andF dependentmonoubiquitinationof FANCD2. W hatelse mightthe FA complexes do in normalcells? Clearly the complex associates with chromatininS-phaseandwhilethereis nobiochemicalevidencethatthisis linkedwitha repair function,it is an appealing assumption in view of the importance of homologous recom-binationrepair in S phase.'I t is also specificallyappealing in light of the linkagebetween the BR CAl/2pathways andthe FA pathway. FA proteins, BR CAl,and BRCA2 are knownto protectcells fromcross-linking agentand oxidationinducedgenotoxicity.'^'^O therproteinsfunctioninthesewaysas well, including ATR, Mrel l,Tip60, NBSl, R ad51, R ad54, Rev3, Snml, X R CC2, X R CC3, ER CCl, BLM,and Xpf,althoughnoneare associated withthedisease Fanconianemia.However,the precise biochemical functionsof the FA complexes are unclear. Based on the current literature, itseemsmostlikelythattheFA proteinscreatescaffoldsforassemblyandproperfoldingof enzymes and multimeric complexes and that the client substrates of the FA proteins eachfunc-tiontoprotectthegenomeandsetthresholdsforresponsestoapoptoticcues.I tiswidely expectedthatthese proteinsare eff^ectorsof DNArepair(because of theirknownfunctionin transcriptioncoupledandhomologousrecombinationrepair,'^'^^^) butbiochemicalstud-ies have yet to confirmthis. Moreover,as a reasonable parallel model, the BR CAlandBRCA2 proteinsexhibitotheractivitiesthantheirroleinmodulationofgenotoxicity.Theydirectly participate in transcriptionalcontrol,^'cytoplasmic signal transduction,andregulation of differentiation.^'^'^ More work needs to be done to clarify the FA-BRCAlrelationshipsin functionalterms. Other Complexes ThecomponentsoftheFA complexesareincreasinginnumberasitbecomesclearthat there is variability frommethod-to-methodand fromone subcellular compartment toanother. Formationof complexesis a dynamic process thatcanbe influencedby cell cycle phase, or by chemicalorbiologicalcues.UsingchromatographicanalysesKupfer s groupdiscoveredthat theFA "core complex" varies