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MolecularHematology Molecular Hematology FOURTHEDITION Editedby DrewProvan MDFRCPFRCPath
EmeritusReaderinAutoimmuneHaematology
DepartmentofHaematology
BartsandTheLondonSchoolofMedicineandDentistry
QueenMaryUniversityofLondon,UK
JohnG.Gribben MDDScFRCPFRCPathFMedSci
ProfessorofMedicalOncology
BartsCancerInstitute
BartsandTheLondonSchoolofMedicineandDentistry
QueenMaryUniversityofLondon,UK
Thiseditionfirstpublished2020 ©2020byJohnWiley&Sons
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LibraryofCongressCataloging-in-PublicationData
Names:Provan,Drew,editor.|Gribben,Johneditor.
Title:Molecularhematology/editedbyDrewProvan,JohnG.Gribben.
Description:Fourthedition.|Hoboken,NJ:Wiley-Blackwell,2020.|Includesbibliographical referencesandindex.
Identifiers:LCCN2019034376(print)|LCCN2019034377(ebook)|ISBN9781119252870(hardback)| ISBN9781119252955(adobepdf)|ISBN9781119252931(epub)
Subjects:MESH:HematologicDiseases|MolecularBiology–methods
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Dedication Wewouldliketodedicatethisbooktotwopeople:ourdearfriendandcolleague,ProfessorSirDavidWeatherall,whosadly passedawayon8December2018.Hewastrulyapioneerofmolecularbiologyandwasthefirstphysicianscientisttouse moleculartechniquestostudyhematologicaldisease.Wewillallmisshimverymuch. Inaddition,wewouldliketodedicatethebooktoValProvan.Alwaysinourthoughtsandmuchmissed.
Contents Contributors, ix
Prefacetothefourthedition, xiii
Furtherreading, xv
Acknowledgments, xvi
1Beginnings:themolecularpathologyofhemoglobin, 1 DavidWeatherall
2Stemcells, 21 DavidT.Scadden
3Thegeneticsofacutemyeloidleukemias, 37 AmyM.Trottier&CarolynJ.Owen
4Moleculardiagnosticsandriskassessmentin myeloidmalignancies, 49 ChristianScharenberg&TorstenHaferlach
5Molecularbasisofacutelymphoblasticleukemia, 59 BelaPatel&FionaFernando
6Chronicmyeloidleukemia, 71 HagopKantarjian,JorgeCortes,EliasJabbour& SusanO’Brien
7Myeloproliferativeneoplasms, 87 JyotiNangalia,AnthonyJ.Bench,AnthonyR. Green&AnnaL.Godfrey
8Lymphomagenetics, 101 JenniferL.Crombie,AnthonyLetai&JohnG.Gribben
9Themolecularbiologyofchroniclymphocytic leukemia, 111 JohnG.Gribben
10Themolecularbiologyofmultiplemyeloma, 121 WeeJooChng&P.LeifBergsagel
11Themolecularbasisofbonemarrowfailure syndromesandredcellenzymopathies, 131 DeenaIskander,LucioLuzzatto&Anastasios Karadimitris
12Anemiaofchronicdisease, 155 TomasGanz
13Themolecularbasisofironmetabolism, 161 NancyC.Andrews&TomasGanz
14Hemoglobinopathiesduetostructuralmutations, 173 D.MarkLayton&StevenOkoli
15Molecularpathogenesisofmalaria, 193 DavidJ.Roberts,ArnabPain&ChetanE.Chitnis
16Molecularcoagulationandthrombophilia, 207 BjornDahlback&AndreasHillarp
17Themolecularbasisofhemophilia, 221 DanielP.Hart&PaulL.F.Giangrande
18ThemolecularbasisofvonWillebranddisease, 235 LucianoBaronciani
19Plateletdisorders, 251 KennethJ.Clemetson
20Themolecularbasisofbloodcellalloantigens, 267 CristinaNavarrete,LouiseTilley,WinnieChong &ColinJ.Brown
21Functionsofbloodgroupantigens, 285 JonathanS.Stamler&MarilynJ.Telen
22Autoimmunehematologicaldisorders, 297 DrewProvan&JohnW.Semple
23Moleculartherapeuticsinhematology:gene therapy, 319 WilliamM.McKillop&JeffreyA.Medin
24Pharmacogenomics, 339 LeoKager&WilliamE.Evans
25Historyanddevelopmentofmolecularbiology, 353 PaulMoss
26Cancerstemcells, 363 SaraAli&DominiqueBonnet
27Molecularbasisoftransplantation, 373 FrancescoDazzi&AntonioGalleu
Index,389
Contributors SaraAliMD
HaematopoieticStemCellLaboratory,TheFrancisCrick Institute,London,UK
NancyC.AndrewsMD,PhD DukeUniversitySchoolofMedicine,Durham,NC,USA
LucianoBaroncianiPhD
FondazioneIRCCSCa’GrandaOspedaleMaggiorePoliclinico,AngeloBianchiBonomiHemophiliaandThrombosis Center,Milan,Italy
AnthonyJ.BenchMA,PhD LaboratoryMedicine,NHSLothian,Edinburgh,UK
P.LeifBergsagelMD
DivisionofHematology-Oncology,ComprehensiveCancer Center,MayoClinicArizona,Scottsdale,AZ,USA
DominiqueBonnetPhD
HaematopoieticStemCellLaboratory,TheFrancisCrick Institute,London,UK
ColinJ.BrownPhD,FRCPath
HistocompatibilityandImmunogeneticsLaboratory,NHS BloodandTransplant;FacultyofLifeSciences&Medicine, King’sCollegeLondon,London,UK
ChetanE.ChitnisMSc,MA,PhD MalariaGroup,PasteurInstitute,Paris,France
WeeJooChngMBChB,MRCP,FRCPath
NationalUniversityCancerInstitute,NationalUniversityHealthSystemofSingapore;UniversityofSingapore, NationalUniversityHospital,Singapore
WinnieChongPhD
HistocompatibilityandImmunogeneticsServiceDevelopmentLaboratory,NHSBloodandTransplant,London,UK
KennethJ.ClemetsonPhD,ScD,CChem,FRSC DepartmentofHaematology,Inselspital,UniversityofBerne, Berne,Switzerland
JorgeCortesMD
DepartmentofLeukemia,UniversityofTexasMDAnderson CancerCenter,Houston,TX,USA
JenniferL.CrombieMD
DepartmentofMedicalOncology,Dana-FarberCancer Institute,HarvardMedicalSchool,Boston,MA,USA
Bj ¨ ornDahlb ¨ ackMD,PhD DepartmentofTranslationalMedicine,SectionofClinical Chemistry,LundUniversity,UniversityHospital,Malmo, Sweden
FrancescoDazziMD,PhD
SchoolofCancer&PharmaceuticalSciences,King’sCollege London;King’sHealthPartnersCancerResearchUKCentre, London,UK
WilliamE.EvansPharmD
StJudeChildren’sResearchHospital,Memphis,TN,USA
FionaFernandoMD
CentreofHaemato-Oncology,BartsCancerInstitute,Queen MaryUniversityofLondon,London,UK
AntonioGalleuMD,PhD
SchoolofCancer&PharmaceuticalSciences,King’sCollege London;King’sHealthPartnersCancerResearchUKCentre, London,UK
TomasGanzPhD,MD
DepartmentofMedicine,DavidGeffenSchoolofMedicine atUCLA,LosAngeles,CA,USA
PaulL.F.GiangrandeMD,FRCP,FRCPath,FRCPCH
FormerlyofOxfordHaemophiliaandThrombosisCentre, ChurchillHospital,Oxford,UK
AnnaL.GodfreyPhD,MRCP,FRCPath DepartmentofHaematology,CambridgeUniversityHospitalsNHSFoundationTrust,Cambridge,UK
AnthonyR.GreenPhD,FRCP,FRCPath,FMedSci DepartmentofHaematology,CambridgeInstituteforMedicalResearch;WellcomeMedicalResearchCouncilStemCell Institute,Cambridge,UK
JohnG.GribbenMD,DSc,FRCP,FRCPath,FMedSci BartsCancerInstitute,BartsandTheLondonSchoolof MedicineandDentistry,QueenMaryUniversityofLondon, London,UK
TorstenHaferlachMD
MLLMunichLeukemiaLaboratory,Munich,Germany
DanielP.HartFRCP,FRCPath,PhD
TheRoyalLondonHospitalHaemophiliaCentre,Bartsand TheLondonSchoolofMedicineandDentistry,QueenMary UniversityofLondon,London,UK
AndreasHillarpPhD
DepartmentofClinicalChemistryandTransfusion Medicine,HallandCountyHospital,Halmstad,Sweden
DeenaIskanderMD,PhD,MRCP CentreforHaematology,ImperialCollegeLondon,HammersmithHospital,London,UK
EliasJabbourMD
DepartmentofLeukemia,UniversityofTexasMDAnderson CancerCenter,Houston,TX,USA
LeoKagerMD
DepartmentofPediatrics,St.AnnaChildren’sHospital, MedicalUniversityVienna,Austria
HagopKantarjianMD DepartmentofLeukemia,UniversityofTexasMDAnderson CancerCenter,Houston,TX,USA
AnastasiosKaradimitrisPhD,MRCP,FRCPath DepartmentofHaematologyandBloodTransfusion, MuhimbiliUniversityCollegeofHealthSciences,Dar-esSalaam,Tanzania
D.MarkLaytonMBBS,FRCP,FRCPCH CenterforHematology,ImperialCollegeLondon,London, UK
AnthonyLetaiMD,PhD DepartmentofMedicalOncology,Dana-FarberCancer Institute,HarvardMedicalSchool,Boston,MA,USA
LucioLuzzattoMD DepartmentofHaematologyandBloodTransfusion, MuhimbiliUniversityCollegeofHealthSciences,Dar-esSalaam,Tanzania
WilliamM.McKillopPhD DepartmentofPediatrics,MedicalCollegeofWisconsin, Milwaukee,WI,USA
JeffreyA.MedinPhD DepartmentsofPediatricsandBiochemistry,MedicalCollegeofWisconsin,Milwaukee,WI,USA
PaulMossMD,PhD SchoolofCancerSciences,UniversityofBirmingham,Birmingham,UK
JyotiNangaliaPhD,MRCP,FRCPath WelcomeSangerInstitute,Hinxton;DepartmentofHaematology,UniversityofCambridge;Wellcome-Medical ResearchCouncilCambridgeStemCellInstitute,Cambridge,UK
CristinaNavarretePhD,FRCPath HistocompatibilityandImmunogeneticsServiceDevelopmentDepartment,NHSBloodandTransplant;Department ofImmunologyandMolecularPathology,UniversityCollege London,London,UK
SusanO’BrienMD DepartmentofLeukemia,UniversityofTexasMDAnderson CancerCenter,Houston,TX,USA
StevenOkoliMBChB,FRCP,FRCPath CenterforHematology,ImperialCollegeLondon,London, UK
CarolynJ.OwenMD,MDres(UK),FRCPC DivisionofHematologyandHematologicalMalignancies, UniversityofCalgary,FoothillsMedicalCentre,Calgary, Canada
ArnabPainPhD BiologicalandEnvironmentalSciencesandEngineering (BESE)Division,KingAbdullahUniversityofScienceand Technology,Jeddah,SaudiArabia;NuffieldDivisionofClinicalLaboratorySciences(NDCLS),TheJohnRadcliffeHospital,UniversityofOxford,Headington,Oxford,UK
BelaPatelMD,FRCPath,MD(res) CentreofHaemato-Oncology,BartsCancerInstitute,Queen MaryUniversityofLondon,London,UK
DrewProvanMD,FRCP,FRCPath BlizardInstitute,BartsandTheLondonSchoolofMedicine andDentistry,QueenMaryUniversityofLondon,London, UK
DavidJ.RobertsDPhil,MRCP,FRCPath NationalHealthServiceBloodandTransplant(Oxford),The JohnRadcliffeHospital,Oxford,UK
DavidT.ScaddenMD DepartmentofStemCellandRegenerativeBiology,Harvard StemCellInstitute,HarvardUniversity;CenterforRegenerativeMedicine,MassachusettsGeneralHospital,Boston,MA, USA
ChristianScharenbergMD,PhD DepartmentofHematology,SkaraborgsHospital,Skovde; DepartmentofCellandMolecularBiology,KarolinskaInstitute,Stockholm,Sweden
JohnW.SemplePhD DivisionofHematologyandTransfusionMedicine,Lund University,Lund,Sweden
JonathanS.StamlerMD
HarringtonDiscoveryInstituteandInstituteofTransformativeMolecularMedicine,UniversityHospitalsCleveland MedicalCenterandCaseWesternReserveUniversity,Cleveland,OH,USA
MarilynJ.TelenMD DepartmentofMedicine,DivisionofHematology,Duke UniversityMedicalCenter,Durham,NC,USA
LouiseTilleyPhD
InternationalBloodGroupReferenceLaboratory,NHS BloodandTransplant,Bristol,UK
AmyM.TrottierMSc,MD,FRCPC DivisionofHematologyandHematologicalMalignancies, UniversityofCalgary,FoothillsMedicalCentre,Calgary, Canada
DavidWeatherallMD,FRCP,FRS FormerlyofWeatherallInstituteofMolecularMedicine,The JohnRadcliffeHospital,Oxford,UK
Prefacetothefourthedition Hematologyisafast-movingdisciplinewithinnovationboth diagnosticallyandtherapeutically.Inthe19yearssincethe firsteditionof MolecularHematology waspublished,many advanceshavebeenmade.Moleculartechniqueshavehelped explainthebasisofmanydiseases,startinginitiallywithred celldisordersandhemostasis.However,thankstotheuseof molecularbiologywecannowdiagnoseandstratifypatients withdiseasessuchasleukemia,myeloma,myeloproliferative neoplasms,andothers.Suchadvancesintechnologyhavenot onlyhelpedexplaintheunderlyingbasisofthediseases,but havealsoprovidedtargetsfortreatment.
Theworldofredcellsstartedthewholespecialtyofmolecularmedicineand,usingmolecularbiologytechniques, manyofthephenotypicfeaturesofredcelldisordershave beenexplained.ThisisdiscussedeloquentlybythelateProfessorSirDavidWeatherallatthebeginningofthebook. Othernon-malignantareaswhichhavebeenupdatedinclude the FunctionsofBloodGroupAntigens, vonWillebrandDisease,and PlateletDisorders
Undoubtedly,hemato-oncologyhasseenthebiggest explosionintermsofunderstandingthemolecularbasisof diseasessuchasleukemias,lymphomas,themyeloprolifer-
ativeneoplasms,myeloma,andmyelodysplasticsyndromes. Thehugearrayofbiologicalmarkersmakesstratificationand treatmentmuchmoresophisticatedthaneverbefore.Allthe chaptersdealingwithmalignantblooddiseaseshavebeen thoroughlyrevisedandbroughtuptodate.
However,despitethegrowingcomplexityintermsof pathogenesis,diagnosis,andmanagementofpatientswith blooddiseases,theethosofthebookremainsthesame–namely,toprovideasuccinctaccountofthemolecularbiologyofhematologicaldiseasewrittenatalevelwhereitshould beofbenefittoboththeseasonedmolecularbiologistand thepracticingclinicianalike.Wehaveretainedtheoriginal structureforthechapters,high-qualityartwork,and Further Reading sectionsinordertomakethebookvisuallyappealing andrelevanttomodernhematologypractice.
Weverymuchhopeyouenjoythiseditionand,asalways, wewelcomeanycommentsorsuggestionsfromreaders, whichwewillattempttoincorporateintothenextedition.
DrewProvan JohnGribben
Furtherreading Anderson,K.C.andNess,P.M.(eds.)(2000). ScientificBasisofTransfusionMedicine:ImplicationsforClinicalPractice,2e.Philadelphia,PA: WBSaunders. Beutler,E.andLichtman,M.A.(eds.). Williams’Hematology,6e.New York:McGraw-Hill.
Cooper,G.M.(1997). TheCell:AMolecularApproach.Washington,DC: ASMPress.
Cox,T.M.andSinclair,J.(1997). MolecularBiologyinMedicine.Oxford: BlackwellScience. Jameson,J.L.(ed.)(1998). PrinciplesofMolecularMedicine.NewYork: HumanaPress.
Mullis,K.B.(1990).Theunusualoriginofthepolymerasechainreaction. ScientificAmerican262:56–65. Roitt,I.(2001). Roitt’sEssentialImmunology,10e.Oxford:BlackwellScience.
Stamatoyannopoulos,G.,Nienhuis,A.W.,Majerus,P.W.,andVarmus, H.(eds.)(2000). TheMolecularBasiseofBloodDiseases,2e.Philadelphia,PA:W.B.Saunders.
Watson,J.D.,Gilman,M.,Witkowski,J.,andZoller,M.(eds.)(1992). RecombinantDNA,2e.NewYork:ScientificAmericanBooks.
Acknowledgments WewouldliketoexpressthankstoClaireBonnett,Publisher,andDeirdreBarry,SeniorEditorialAssistant,fortheirhelpwith thiswork.
Chapter1 Beginnings:themolecular pathologyofhemoglobin DavidWeatherall
WeatherallInstituteofMolecularMedicine,JohnRadcliffeHospital,Oxford,UK
Historicalbackground, 1
Thestructure,geneticcontrol,andsynthesisofnormalhemoglobin, 2
Themolecularpathologyofhemoglobin, 6
Genotype–phenotyperelationshipsinthethalassemias, 12
Structuralhemoglobinvariants, 16
Historicalbackground LinusPaulingfirstusedtheterm“moleculardisease”in1949, afterthediscoverythatthestructureofsicklecellhemoglobin differedfromthatofnormalhemoglobin.Indeed,itwasthis seminalobservationthatledtotheconceptof molecular medicine,thedescriptionofdiseasemechanismsatthelevel ofcellsandmolecules.However,untilthedevelopmentof recombinantDNAtechnologyinthemid-1970s,knowledge ofeventsinsidethecellnucleus,notablyhowgenesfunction, couldonlybethesubjectofguessworkbasedonthestructure andfunctionoftheirproteinproducts.However,assoonas itbecamepossibletoisolatehumangenesandtostudytheir properties,thepicturechangeddramatically.
Progressoverthelast30yearshasbeendrivenbytechnologicaladvancesinmolecularbiology.Atfirstitwaspossible onlytoobtainindirectinformationaboutthestructureand functionofgenesbyDNA/DNAandDNA/RNAhybridization;thatis,byprobingthequantityorstructureofRNA orDNAbyannealingreactionswithmolecularprobes.The nextmajoradvancewastheabilitytofractionateDNAinto piecesofpredictablesizewithbacterialrestrictionenzymes. Thisledtotheinventionofatechniquethatplayedacentral roleintheearlydevelopmentofhumanmoleculargenetics, called Southernblotting afterthenameofitsdeveloper,Edwin Southern.Thismethodallowedthestructureandorganizationofgenestobestudieddirectlyforthefirsttimeandled tothedefinitionofanumberofdifferentformsofmolecular pathology.
OnceitwaspossibletofractionateDNA,itsoonbecame feasibletoinsertthepiecesintovectorsabletodivide
Molecularaspectsofthehighfrequencyofthehemoglobinvariants, 17
Molecularaspectsofthepreventionandmanagementofthe hemoglobindisorders, 18
Postscript, 18
Furtherreading, 18
withinbacteria.Thesteadyimprovementinthepropertiesofcloningvectorsmadeitpossibletogeneratelibraries ofhumanDNAgrowinginbacterialcultures.Ingenious approachesweredevelopedtoscanthelibrariestodetect genesofinterest;oncepinpointed,theappropriatebacterialcoloniescouldbegrowntogeneratelargerquantitiesof DNAcarryingaparticulargene.Lateritbecamepossible tosequencethesegenes,persuadethemtosynthesizetheir productsinmicroorganisms,culturedcells,orevenother species,andhencetodefinetheirkeyregulatoryregions.
Theearlyworkinthefieldofhumanmoleculargeneticsfocusedondiseasesinwhichtherewassomeknowledgeofthegeneticdefectattheproteinorbiochemicallevel. However,oncelinkagemapsofthehumangenomebecame available,followingtheidentificationofhighlypolymorphic regionsofDNA,itwaspossibletosearchforanygenefora disease,evenwherethecausewascompletelyunknown.This approach,firstcalled reversegenetics andlaterrechristened positionalcloning,ledtothediscoveryofgenesformany importantdiseases.
Asmethodsforsequencingwereimprovedandautomated, thoughtsturnedtothenextmajorgoalinthisfield,whichwas todeterminethecompletesequenceofthebasesthatconstituteourgenesandallthatliesbetweenthem:theHuman GenomeProject.Thisremarkableendeavorwasfinallycompletedin2006.Thefurtherunderstandingofthefunctions andregulationofourgeneswillrequiremultidisciplinary researchencompassingmanydifferentfields.Thenextstage intheHumanGenomeProject,called genomeannotation, entailsanalyzingtherawDNAsequenceinordertodetermineitsbiologicalsignificance.Oneofthemainventures intheeraoffunctionalgenomicswillbeinwhatistermed proteomics,thelarge-scaleanalysisoftheproteinproductsof genes.Theultimategoalwillbetotrytodefinetheprotein
complement,orproteome,ofcellsandhowthemanydifferentproteinsinteractwithoneanother.Tothisend,largescalefacilitiesarebeingestablishedforisolatingandpurifyingtheproteinproductsofgenesthathavebeenexpressed inbacteria.Theirstructurecanthenbestudiedbyavarietyofdifferenttechniques,notablyX-raycrystallography andnuclearmagneticresonancespectroscopy.Thecrystallographicanalysisofproteinsisbeinggreatlyfacilitated bytheuseofX-raybeamsfromasynchrotronradiation source.
Inthelastfewyearsboththeutilityandextremecomplexityofthefruitsofthegenomeprojecthavebecome apparent.Theexistenceofthousandsofsingle-nucleotide polymorphisms(SNPs)hasmadeitpossibletosearchfor genesofbiologicalormedicalsignificance.Thediscoveryof familiesofregulatoryRNAsandproteinsisstartingtoshed lightonhowthefunctionsofthegenomearecontrolled, andstudiesofacquiredchangesinitsstructure, epigenetics, promisetoprovidesimilarinformation.Recentdevelopmentsinnew-generationsequencingofDNAandRNAare alsoprovidinginvaluableinformationaboutmanyaspects ofgeneregulation.
Duringthisremarkableperiodoftechnicaladvance,considerableprogresshasbeenmadetowardanunderstanding ofthepathologyofdiseaseatthemolecularlevel.Thishas hadaparticularimpactonhematology,leadingtoadvances intheunderstandingofgenefunctionanddiseasemechanismsinalmosteveryaspectofthefield.
Theinheriteddisordersofhemoglobin–thethalassemias andstructuralhemoglobinvariants,thecommonesthuman monogenicdiseases–werethefirsttobestudiedsystematicallyatthemolecularlevelandagreatdealisknownabout theirgenotype–phenotyperelationships.Thisfieldledthe waytomolecularhematologyand,indeed,tothedevelopmentofmolecularmedicine.Thus,eventhoughthegeneticsofhemoglobiniscomplicatedbythefactthatdifferent varietiesareproducedatparticularstagesofhumandevelopment,themolecularpathologyofthehemoglobinopathies providesanexcellentmodelsystemforunderstandingany monogenicdiseaseandthecomplexinteractionsbetween genotypeandenvironmentthatunderliemanymultigenic disorders.
InthischapterIconsiderthestructure,synthesis,and geneticcontrolofthehumanhemoglobins,describethe molecularpathologyofthethalassemias,anddiscussbriefly howthecomplexinteractionsoftheirdifferentgenotypes producearemarkablydiversefamilyofclinicalphenotypes; thestructuralhemoglobinvariantsarediscussedinmore detailinChapter14.Readerswhowishtolearnmoreabout themethodsofmoleculargenetics,particularlyasappliedto thestudyofhemoglobindisorders,arereferredtothereviews citedattheendofthischapter.
Thestructure,geneticcontrol,and synthesisofnormalhemoglobin Structureandfunction Thevaryingoxygenrequirementsduringembryonic,fetal, andadultlifearereflectedinthesynthesisofdifferent structuralhemoglobinsateachstageofhumandevelopment. However,theyallhavethesamegeneraltetramericstructure,consistingoftwodifferentpairsofglobinchains,each attachedtoonehememolecule.Adultandfetalhemoglobins have α chainscombinedwith β chains(HbA, α2 β2 ), δ chains(HbA2 , α2 δ2 ),and γ chains(HbF, α2 γ2 ).In embryos, α-likechainscalled ζ chainscombinewith γ chainstoproduceHbPortland(ζ2 γ2 ),orwith ε chainsto makeHbGower1(ζ2 ε2 ),while α and ε chainsformHb Gower2(α2 ε2 ).Fetalhemoglobinisheterogeneous;there aretwovarietiesof γ chainthatdifferonlyintheiramino acidcompositionatposition136,whichmaybeoccupiedby eitherglycineoralanine; γ chainscontainingglycineatthis positionarecalled G γ chains,thosewithalanine A γ chains (Figure1.1).
Thesynthesisofhemoglobintetramersconsistingoftwo unlikepairsofglobinchainsisabsolutelyessentialforthe effectivefunctionofhemoglobinasanoxygencarrier.The classicalsigmoidshapeoftheoxygendissociationcurve, whichreflectstheallostericpropertiesofthehemoglobin molecule,ensuresthat,athighoxygentensionsinthelungs, oxygenisreadilytakenupandlaterreleasedeffectivelyatthe lowertensionsencounteredinthetissues.Theshapeofthe curveisquitedifferenttothatofmyoglobin,amoleculethat consistsofasingleglobinchainwithhemeattachedtoit, which,likeabnormalhemoglobinsthatconsistofhomotetramersoflikechains,hasahyperbolicoxygendissociation curve.
Thetransitionfromahyperbolictoasigmoidoxygen dissociationcurve,whichisabsolutelycriticalfornormal oxygendelivery,reflectscooperativitybetweenthefour hememoleculesandtheirglobinsubunits.Whenoneof themtakesonoxygen,theaffinityoftheremainingthree increasesmarkedly;thishappensbecausehemoglobincan existintwoconfigurations,deoxy(T)andoxy(R),whereT andRrepresentthetightandrelaxedstates,respectively. TheTconfigurationhasaloweraffinitythantheRfor ligandssuchasoxygen.Atsomepointduringtheaddition ofoxygentothehemes,thetransitionfromtheTtotheR configurationoccursandtheoxygenaffinityofthepartially ligandedmoleculeincreasesdramatically.Theseallosteric changesresultfrominteractionsbetweentheironofthe hemegroupsandvariousbondswithinthehemoglobin tetramer,whichleadtosubtlespatialchangesasoxygenis takenonorgivenup.
Embryo
Fetus Adult
Fig.1.1Thegeneticcontrolofhumanhemoglobinproductioninembryonic,fetal,andadultlife. Thestandardnamesforthesegenes areasfollows:Alphagenes HBA1 and HBA2,Betagene HBB,Gammagenes HBG1 and HBG2,Deltagene HBD,andtheembryonicgenes HBE1 and HBZ
Theprecisetetramericstructuresofthedifferenthuman hemoglobins,whichreflecttheprimaryaminoacid sequencesoftheirindividualglobinchains,arealsovital forthevariousadaptivechangesthatarerequiredtoensure adequatetissueoxygenation.Thepositionoftheoxygen dissociationcurvecanbemodifiedinseveralways.For example,oxygenaffinitydecreaseswithincreasingCO2 tension(theBohreffect).Thisfacilitatesoxygenloadingtothe tissues,whereadropinpHduetoCO2 influxlowersoxygen affinity;theoppositeeffectoccursinthelungs.Oxygenaffinityisalsomodifiedbythelevelof2,3-diphosphoglycerate (2,3-BPG)intheredcell.Increasingconcentrationsshift theoxygendissociationcurvetotheright(i.e.theyreduce oxygenaffinity),whilediminishingconcentrationshavethe oppositeeffect.2,3-BPGfitsintothegapbetweenthetwo β chainswhenitwidensduringdeoxygenation,andinteracts withseveralspecificbindingsitesinthecentralcavityof themolecule.Inthedeoxyconfigurationthegapbetween thetwo β chainsnarrowsandthemoleculecannotbe accommodated.Withincreasingconcentrationsof2,3-BPG, whicharefoundinvarioushypoxicandanemicstates, morehemoglobinmoleculestendtobeheldinthedeoxy configurationandtheoxygendissociationcurveistherefore shiftedtotheright,withmoreeffectivereleaseofoxygen.
Fetalredcellshavegreateroxygenaffinitythanadult redcells,although,interestingly,purifiedfetalhemoglobin hasanoxygendissociationcurvesimilartothatofadult hemoglobin.Thesedifferences,whichareadaptedtotheoxygenrequirementsoffetallife,reflecttherelativeinabilityof HbFtointeractwith2,3-BPGcomparedwithHbA.Thisis becausethe γ chainsofHbFlackspecificbindingsitesfor 2,3-BPG.
Inshort,oxygentransportcanbemodifiedbyavariety ofadaptivefeaturesintheredcellthatincludeinteractions betweenthedifferenthememolecules,theeffectsofCO2 ,and differentialaffinitiesfor2,3-BPG.Thesechanges,together withmoregeneralmechanismsinvolvingthecardiorespiratorysystem,providethemainbasisforphysiologicaladaptationtoanemia.
Geneticcontrolofhemoglobin The α-and β-likeglobinchainsaretheproductsoftwodifferentgenefamilieswhicharefoundondifferentchromosomes(Figure1.1).The β-likeglobingenesformalinked clusteronchromosome11,spreadoverapproximately60kb (kilobaseor1000nucleotidebases).Thedifferentgenesthat formthisclusterarearrangedintheorder5′ –ε–G γ–A γ–ψβ–δ–β–3′ .The α-likegenesalsoformalinkedcluster,in thiscaseonchromosome16,intheorder5′ –ζ–ψζ–ψα1–α2–α1–3′ .The ψβ, ψζ,and ψα genesarepseudogenes;that is,theyhavestrongsequencehomologywiththe β, ζ,and α genes,butcontainanumberofdifferencesthatprevent themfromdirectingthesynthesisofanyproducts.Theymay reflectremnantsofgenesthatwerefunctionalatanearlier stageofhumanevolution.
Thestructureofthehumanglobingenesis,inessence, similartothatofallmammaliangenes.Theyconsistoflong stringsofnucleotidesthataredividedintocodingregions, orexons,andnon-codinginsertscalled interveningsequences (IVSs)orintrons.The β-likeglobingenescontaintwo introns,oneof122–130basepairsbetweencodons30and31 andoneof850–900basepairsbetweencodons104and105 (theexoncodonsarenumberedsequentiallyfromthe5′ to
the3′ endofthegene,i.e.fromlefttoright).Similar,though smaller,intronsarefoundinthe α and ζ globingenes.These intronsandexons,togetherwithshortnon-codingsequences atthe5′ and3′ endsofthegenes,representthemajorfunctionalregionsoftheparticulargenes.However,therearealso extremelyimportantregulatorysequenceswhichsubserve thesefunctionsthatlieoutsidethegenesthemselves.
Atthe5′ non-coding(flanking)regionsoftheglobin genes,asinallmammaliangenes,thereareblocksof nucleotidehomology.Thefirst,theATAbox,isabout30 basesupstream(totheleft)oftheinitiationcodon;thatis,the startwordforthebeginningofproteinsynthesis(seelater). Thesecond,theCCAATbox,isabout70basepairsupstream fromthe5′ endofthegenes.About80–100basesfurther upstreamthereisthesequenceGGGGTG,orCACCC,which maybeinvertedorduplicated.Thesethreehighlyconserved DNAsequences,called promoterelements,areinvolvedin theinitiationoftranscriptionoftheindividualgenes.Finally, inthe3′ non-codingregionofalltheglobingenesthere isthesequenceAATAAA,whichisthesignalforcleavage andpolyAadditiontoRNAtranscripts(seeGeneActionand GlobinSynthesis).
Theglobingeneclustersalsocontainseveralsequences thatconstituteregulatoryelements,whichinteracttopromoteerythroid-specificgeneexpressionandcoordination ofthechangesinglobingeneactivityduringdevelopment.Theseincludetheglobin genesthemselvesandtheir
promoterelements:enhancers(regulatorysequencesthat increasegeneexpressiondespitebeinglocatedataconsiderabledistancefromthegenes)and“master”regulatory sequencescalled,inthecaseofthe β globingenecluster,the locuscontrolregion (LCR)and,inthecaseofthe α genes, HS40(anuclease-hypersensitivesiteinDNA40kbfromthe α globingenes).Eachofthesesequenceshasamodularstructuremadeupofanarrayofshortmotifsthatrepresentthe bindingsitesfortranscriptionalactivatorsorrepressors.
Geneactionandglobinsynthesis TheflowofinformationbetweenDNAandproteinissummarizedinFigure1.2.Whenaglobingeneistranscribed, messengerRNA(mRNA)issynthesizedfromoneofits strands,aprocesswhichbeginswiththeformationofatranscriptioncomplexconsistingofavarietyofregulatoryproteinstogetherwithanenzymecalledRNApolymerase(see later).TheprimarytranscriptisalargemRNAprecursor whichcontainsbothintronandexonsequences.Whilein thenucleus,thismoleculeundergoesavarietyofmodifications.First,theintronsareremovedandtheexonsarespliced together.Theintron/exonjunctionsalwayshavethesame sequence:GTattheir5′ end,andAGattheir3′ end.This appearstobeessentialforaccuratesplicing;ifthereisamutationatthesesitesthisprocessdoesnotoccur.Splicingreflects acomplexseriesofintermediarystagesandtheinteraction
Fig.1.2
ofanumberofdifferentnuclearproteins.Aftertheexonsare joined,themRNAsaremodifiedandstabilized;attheir5′ end acomplexCAPstructureisformed,whileattheir3′ enda stringofadenylicacidresidues(polyA)isadded.ThemRNA processedinthiswaymovesintothecytoplasm,whereit actsasatemplateforglobinchainproduction.Becauseof therulesofbasepairing–thatis,cytosinealwayspairswith thymine,andguaninewithadenine–thestructureofthe mRNAreflectsafaithfulcopyoftheDNAcodonsfromwhich itissynthesized;theonlydifferenceisthat,inRNA,uracil(U) replacesthymine(T).
AminoacidsaretransportedtothemRNAtemplateon carrierscalledtransferRNAs(tRNAs);therearespecific tRNAsforeachaminoacid.Furthermore,becausethegenetic codeisredundant(i.e.morethanonecodoncanencodea particularaminoacid),forsomeoftheaminoacidsthereare severaldifferentindividualtRNAs.Theirorderintheglobin chainisdeterminedbytheorderofcodonsinthemRNA. ThetRNAscontainthreebases,whichtogetherconstitutean anticodon;theseanticodonsarecomplementarytomRNA codonsforparticularaminoacids.Theycarryaminoacids tothetemplate,wheretheyfindtheappropriatepositioning bycodon–anticodonbasepairing.WhenthefirsttRNAis inposition,aninitiationcomplexisformedbetweenseveral proteininitiationfactorstogetherwiththetwosubunitsthat constitutetheribosomes.AsecondtRNAmovesinalongsideandthetwoaminoacidsthattheyarecarryingform apeptidebondbetweenthem;theglobinchainisnowtwo aminoacidresidueslong.Thisprocessiscontinuedalong
themRNAfromlefttoright,andthegrowingpeptidechain istransferredfromoneincomingtRNAtothenext;thatis, themRNAistranslatedfrom5′ to3′ .Duringthistimethe tRNAsareheldinappropriatestericconfigurationwiththe mRNAbythetworibosomalsubunits.Therearespecific initiation(AUG)andtermination(UAA,UAG,andUGA) codons.Whentheribosomesreachtheterminationcodon, translationceases,thecompletedglobinchainsarereleased, andtheribosomalsubunitsarerecycled.Individualglobin chainscombinewithheme,whichhasbeensynthesized throughaseparatepathway,andtheninteractwithonelike chainandtwounlikechainstoformacompletehemoglobin tetramer.
Regulationofhemoglobinsynthesis Theregulationofglobingeneexpressionismediatedmainly atthetranscriptionallevel,withsomefinetuningduring translationandpost-translationalmodificationofthegene products.DNAthatisnotinvolvedintranscriptionisheld tightlypackagedinacompact,chemicallymodifiedformthat isinaccessibletotranscriptionfactorsandpolymerasesandis heavilymethylated.Activationofaparticulargeneisreflected bychangesinthestructureofthesurroundingchromatin, whichcanbeidentifiedbyenhancedsensitivitytonucleases. Erythroidlineage-specificnuclease-hypersensitivesitesare foundatseverallocationsinthe β globingenecluster.Four aredistributedover20kbupstreamfromthe ε globingene intheregionofthe β globinLCR(Figure1.3).Thisvital
Chromosome 11
Chromosome 16
Fig.1.3Thepositionsofthemajorregulatoryregionsinthe β and α globingeneclusters. Thearrowsindicatethepositionofthe erythroidlineage-specificnuclease-hypersensitivesites.HS,hypersensitive.
regulatoryregionisabletoestablishatranscriptionallyactive domainspanningtheentire β globingenecluster.Several enhancersequenceshavebeenidentifiedinthiscluster.A varietyofregulatoryproteinsbindtotheLCR,andtothe promoterregionsoftheglobingenesandtotheenhancer sequences.ItisthoughtthattheLCRandotherenhancer regionsbecomeopposedtothepromoterstoincrease therateoftranscriptionofthegenestowhichtheyare related.
Theseregulatoryregionscontainsequencemotifsfor variousubiquitousanderythroid-restrictedtranscription factors.Bindingsitesforthesefactorshavebeenidentifiedin eachoftheglobingenepromotersandatthehypersensitive siteregionsofthevariousregulatoryelements.Anumber ofthefactorswhichbindtotheseareasarefoundinallcell types.TheyincludeSp1,Yy1,andUsf.Incontrast,anumber oftranscriptionfactorshavebeenidentified,including GATA-1,EKLF,andNF-E2,whicharerestrictedintheir distributiontoerythroidcellsand,insomecases,megakaryocytes,andmastcells.Theoverlappingoferythroid-specific andubiquitous-factorbindingsitesinseveralcasessuggests thatcompetitivebindingmayplayanimportantpartin theregulationoferythroid-specificgenes.Anotherbinding factor,SSP,thestageselectorprotein,appearstointeract specificallywith ε and γ genes.Severalelementsinvolving thechromatinandhistoneacetylationrequiredforaccessof theseregulatoryproteinshavebeenidentified.
Thebindingofhematopoietic-specificfactorsactivatesthe LCR,whichrenderstheentire β globingeneclustertranscriptionallyactive.Thesefactorsalsobindtotheenhancer andpromotersequences,whichworkintandemtoregulatetheexpressionoftheindividualgenesintheclusters.It islikelythatsomeofthetranscriptionalfactorsaredevelopmentalstagespecific,andhencemayberesponsiblefor thedifferentialexpressionoftheembryonic,fetal,andadult globingenes.The α globingeneclusteralsocontainsanelement,HS40,whichhassomestructuralfeaturesincommon withthe β LCR,althoughitisdifferentinaspectsofitsstructure.Anumberofenhancer-likesequenceshavebeenidentified,althoughitisbecomingclearthattherearefundamental differencesinthepatternofregulationofthetwoglobingene clusters.
Inadditiontothedifferentregulatorysequencesoutlined, therearealsosequenceswhichmaybeinvolvedspecifically with“silencing”ofgenes,notablythosefortheembryonic hemoglobins,duringdevelopment.
Somedegreeofregulationismediatedbydifferencesin theratesofinitiationandtranslationofthedifferentmRNAs, andatthepost-transcriptionallevelbydifferentialaffinity fordifferentproteinsubunits.However,thiskindofposttranscriptionalfinetuningprobablyplaysarelativelysmall roleindeterminingtheoveralloutputoftheglobingene products.
Regulationofdevelopmentalchanges inglobingeneexpression Duringdevelopment,thesiteofredcellproductionmoves fromtheyolksactothefetalliverandspleen,andthence tobonemarrowintheadult.Embryonic,fetal,andadult hemoglobinsynthesisisapproximatelyrelatedintimeto thesechangesinthesiteoferythropoiesis,althoughitisquite clearthatthevariousswitches,betweenembryonicandfetal andbetweenfetalandadulthemoglobinsynthesis,arebeautifullysynchronizedthroughoutthesedifferentsites.Fetal hemoglobinsynthesisdeclinesduringthelatermonthsof gestation,andHbFisreplacedbyHbAandHbA2 bythe endofthefirstyearoflife.
Althoughtheexactmechanismoftheswitchfromfetalto adulthemoglobinisstillnotunderstood,recentstudiesof patientswithunusuallyhighlevelsofHbFandgenome-wide associationstudies(GWAS)haveyieldedextremelypromisinginformationaboutsomeoftheregulatorygenesinvolved. TheyincludeBCL11A,MYB,andKLF1.Itisclearfromstudiesoftheseandrelatedgenesthattheyareinvolveddirectlyin theregulationofhemoglobinswitching,workwhichisyieldinggreatpromiseforthedevelopmentoffuturetechnology forincreasingHbFsynthesistomodifythephenotypeofthalassemiasandsicklecellanemia.
Themolecularpathology ofhemoglobin Asisthecaseformanymonogenicdiseases,theinheriteddisordersofhemoglobinfallintotwomajorclasses.First,there arethosethatresultfromreducedoutputofoneorother globingenes,the thalassemias.Second,thereisawiderange ofconditionsthatresultfromtheproductionof structurally abnormalglobinchains;thetypeofdiseasedependsonhow theparticularalterationinproteinstructureinterfereswith itsstabilityorfunction.Ofcourse,nobiologicalclassificationisentirelysatisfactoryandthosewhichattempttodefine thehemoglobindisordersarenoexception.Therearesome structuralhemoglobinvariantswhichhappentobesynthesizedatareducedrateandhenceareassociatedwithaclinicalpicturesimilartothalassemia.Andthereareotherclasses ofmutationswhichsimplyinterferewiththenormaltransitionfromfetaltoadulthemoglobinsynthesis,afamilyof conditionsgiventhegeneraltitle hereditarypersistenceoffetal hemoglobin (HPFH).Furthermore,becausethesediseasesare allsocommonandoccurtogetherinparticularpopulations, itisnotuncommonforanindividualtoinheritagenefor oneorotherformofthalassemiaandastructuralhemoglobin variant.Theheterogeneousgroupofconditionsthatresults fromthesedifferentmutationsandinteractionsissummarizedinTable1.1.
Table1.1 Thethalassemiasandrelateddisorders
α Thalassemia γ Thalassemia
α0
α+ δ Thalassemia
Deletion( α)
Non-deletion(αT ) εγδβ Thalassemia
β Thalassemia Hereditarypersistenceof β0 fetalhemoglobin
β+ Deletion
NormalHbA2 (δβ)0
“Silent” Non-deletion
Dominant Linkedto β globingenes
δβ Thalassemia G γβ+ (δβ)+A γβ+ (δβ)0 Unlinkedto β globingenes (A γδβ)0
Overrecentyears,determinationofthemolecularpathologyofthetwocommonformsofthalassemia, α and β,has providedaremarkablepictureoftherepertoireofmutations thatcanunderliehumanmonogenicdisease.Inthesections thatfollowIdescribe,inoutline,thedifferentformsofmolecularpathologythatunderlietheseconditions.
The β thalassemias
Therearetwomainclassesof β thalassemia, β0 thalassemia, inwhichthereisanabsenceof β globinchainproduction, and β+ thalassemia,inwhichthereisavariablereduction intheoutputof β globinchains.AsshowninFigure1.4, mutationsofthe β globingenesmaycauseareducedoutput ofgeneproductattheleveloftranscriptionormRNA processing,ortranslation,orthroughthestabilityofthe globingeneproduct.
Defective β globingenetranscription Thereareavarietyofmechanismsthatinterferewithnormaltranscriptionofthe β globingenes.First,thegenesmay beeithercompletelyorpartiallydeleted.Overall,deletions ofthe β globingenesarenotcommonlyfoundinpatients with β thalassemia,withoneexception:a619-bpdeletion involvingthe3′ endofthegeneisfoundfrequentlyinthe SindpopulationsofIndiaandPakistan,whereitconstitutes about30%ofthe β thalassemiaalleles.Otherdeletionsare extremelyrare.
Amuchmorecommongroupofmutations,whichresults inamoderatedecreaseintherateoftranscriptionofthe β globingenes,involvessingle-nucleotidesubstitutionsin orneartheTATAboxatabout 30nucleotides(nt)from thetranscriptionstartsite,orintheproximalordistalpromoterelementsat 90and 105nt.Thesemutationsresult indecreased β globinmRNAproduction,rangingfrom10% to25%ofthenormaloutput.Thus,theyareusuallyassociatedwiththemildformsof β+ thalassemia.TheyareparticularlycommoninAfricanpopulations,anobservationwhich explainstheunusualmildnessof β thalassemiainthisracial group.Oneparticularmutation,C → Tatposition 101nt tothe β globingene,causesanextremelymilddeficitof β globinmRNA.Indeed,thisalleleissomildthatitiscompletelysilentincarriersandcanonlybeidentifiedbyitsinteractionwithmoresevere β thalassemiaallelesincompound heterozygotes.
Mutationsthatcauseabnormalprocessing ofmRNA Asmentionedearlier,theboundariesbetweenexonsand intronsaremarkedbytheinvariantdinucleotidesGTatthe donor(5′ )siteandAGattheacceptor(3′ )site.Mutationsthat affecteitherofthesesitescompletelyabolishnormalsplicing
Point mutations
Fig.1.4Themutationsofthe β globingenethatunderlie β thalassemia. Theheavyblacklinesindicatethelengthofthedeletions.The pointmutationsaredesignatedasfollows:PR,promoter;C,CAPsite;I,initiationcodon;FS,frameshiftandnonsensemutations;SPL,splice mutations;PolyA,polyAadditionsitemutations.
Deletions
andproducethephenotypeof β0 thalassemia.Thetranscriptionofgenescarryingthesemutationsappearstobenormal, butthereiscompleteinactivationofsplicingatthealtered junction.
Anotherfamilyofmutationsinvolveswhatarecalled splice siteconsensussequences.AlthoughonlytheGTdinucleotide isinvariantatthedonorsplicesite,thereisconservation ofadjacentnucleotidesandacommon,orconsensus, sequenceoftheseregionscanbeidentified.Mutations withinthissequencecanreducetheefficiencyofsplicing tovaryingdegrees,becausetheyleadtoalternatesplicing atthesurroundingcrypticsites.Forexample,mutationsof thenucleotideatposition5ofIVS-1(thefirstintervening sequence),G → CorT,resultinamarkedreductionof β chainproductionandinthephenotypeofsevere β+ thalassemia.Ontheotherhand,thesubstitutionofCforT atposition6inIVS-1leadstoonlyamildreductioninthe outputof β chains.
Anothermechanismthatleadstoabnormalsplicing involves crypticsplicesites.TheseareregionsofDNAwhich, ifmutated,assumethefunctionofasplicesiteataninappropriateregionofthemRNAprecursor.Forexample,avariety ofmutationsactivateacrypticsitewhichspanscodons24–27ofexon1ofthe β globingene.ThissitecontainsaGT dinucleotide,andadjacentsubstitutionsthatalteritsothatit morecloselyresemblestheconsensusdonorsplicesiteresult initsactivation,eventhoughthenormalsplicesiteisintact.A mutationatcodon24GGT → GGA,thoughitdoesnotalter theaminoacidwhichisnormallyfoundinthispositionin the β globinchain(glycine),allowssomesplicingtooccur atthissiteinsteadoftheexon–intronboundary.Thisresults intheproductionofbothnormalandabnormallyspliced β
globinmRNAandhenceintheclinicalphenotypeofsevere β thalassemia.Interestingly,mutationsatcodons19,26,and 27resultinbothreducedproductionofnormalmRNA(due toabnormalsplicing)andanaminoacidsubstitutionwhen themRNAwhichissplicednormallyistranslatedintoprotein.TheabnormalhemoglobinsproducedareHbMalay,Hb E,andHbKnossos,respectively.Allthesevariantsareassociatedwithamild β+ thalassemia-likephenotype.Thesemutationsillustratehowsequencechangesincodingratherthan IVSinfluenceRNAprocessing,andunderlinetheimportanceofcompetitionbetweenpotentialsplicesitesequences ingeneratingbothnormalandabnormalvarietiesof β globin mRNA.
Crypticsplicesitesinintronsmayalsocarrymutations thatactivatethemeventhoughthenormalsplicesitesremain intact.AcommonmutationofthiskindinMediterranean populationsinvolvesabasesubstitutionatposition110in IVS-1.Thisregioncontainsasequencesimilartoa3′ acceptorsite,thoughitlackstheinvariantAGdinucleotide.The changeoftheGtoAatposition110createsthisdinucleotide. Theresultisthatabout90%oftheRNAtranscriptsplicesto thisparticularsiteandonly10%tothenormalsite,againproducingthephenotypeofsevere β+ thalassemia(Figure1.5). Severalother β thalassemiamutationshavebeendescribed whichgeneratenewdonorsiteswithinIVS-2ofthe β globin gene.
Anotherfamilyofmutationsthatinterfereswith β globin geneprocessinginvolvesthesequenceAAUAAAinthe3′ untranslatedregions,whichisthesignalforcleavageand polyadenylationofthe β globingenetranscript.Somehow, thesemutationsdestabilizethetranscript.Forexample,a T → Csubstitutioninthissequenceleadstoonlyone-tenthof
Fig.1.5Thegenerationofanewsplicesitein anintronasthemechanismforaformof β+ thalassemia.Fordetailsseetext.
Normal splicing
thenormalamountof β globinmRNAtranscript,andhence tothephenotypeofamoderatelysevere β+ thalassemia. Anotherexampleofamutationwhichprobablyleadsto defectiveprocessingofthefunctionof β globinmRNAisthe single-basesubstitutionA → CintheCAPsite.Itisnotyet understoodhowthismutationcausesareducedrateoftranscriptionofthe β globingene.
Thereisanothersmallsubsetofraremutationsthatinvolve the3′ untranslatedregionofthe β globingene,andtheseare associatedwithrelativelymildformsof β thalassemia.Itis thoughtthattheseinterfereinsomewaywithtranscription, butthemechanismisunknown.
Mutationsthatresultinabnormal translationof β globinmRNA
Therearethreemainclassesofmutationsofthiskind. Basesubstitutionsthatchangeanaminoacidcodontoa chainterminationcodonpreventthetranslationof β globin mRNAandresultinthephenotypeof β0 thalassemia.Several mutationsofthiskindhavebeendescribed;thecommonest,involvingcodon17,occurswidelythroughoutSoutheastAsia.Similarly,acodon39mutationisencounteredfrequentlyintheMediterraneanregion.
Thesecondclassinvolvestheinsertionordeletionof one,two,orfournucleotidesinthecodingregionofthe β globingene.Thesedisruptthenormalreadingframe,cause aframeshift,andhenceinterferewiththetranslationof β globinmRNA.Theendresultistheinsertionofanomalous aminoacidsaftertheframeshiftuntilaterminationcodon isreachedinthenewreadingframe.Thistypeofmutation alwaysleadstothephenotypeof β0 thalassemia.
Finally,thereareseveralmutationswhichinvolvethe β globingeneinitiationcodonandwhich,presumably,reduce theefficiencyoftranslation.
Unstable β globinchainvariants Someformsof β thalassemiaresultfromthesynthesisof highlyunstable β globinchainswhichareincapableofforminghemoglobintetramersandwhicharerapidlydegraded, leadingtothephenotypeof β0 thalassemia.Indeed,inmany oftheseconditionsnoabnormalglobinchainproductcanbe demonstratedbyproteinanalysis,andthemolecularpathologyhastobeinterpretedsimplyonthebasisofaderived sequenceofthevariant β chainobtainedbyDNAanalysis. Recentstudieshaveprovidedsomeinterestinginsights intohowcomplexclinicalphenotypesmayresultfromthe synthesisofunstable β globinproducts.Forexample,there isaspectrumofdisordersthatresultfrommutationsinexon 3whichgiverisetoamoderatelysevereformof β thalassemiainheterozygotes.Ithasbeenfoundthatnonsenseor frameshiftmutationsinexonsIandIIareassociatedwiththe
absenceofmRNAfromthecytoplasmofredcellprecursors. Thisappearstobeanadaptivemechanism,called nonsensemediateddecay,wherebyabnormalmRNAofthistypeisnot transportedtothecytoplasm,whereitwouldactasatemplatefortheproductionoftruncatedgeneproducts.However,inthecaseofexonIIImutations,apparentlybecause thisprocessrequiresthepresenceofanintactupstreamexon, theabnormalmRNAistransportedintothecytoplasmand hencecanactasatemplatefortheproductionofunstable β globinchains.Thelatterprecipitateintheredcellprecursorstogetherwithexcess α chainstoformlargeinclusionbodies,andhencethereisenoughglobinchainimbalanceinheterozygotestoproduceamoderatelyseveredegree ofanemia.
The α thalassemias Themolecularpathologyofthe α thalassemiasismorecomplicatedthanthatofthe β thalassemias,simplybecausethere aretwo α globingenesperhaploidgenome.Thus,thenormal α globingenotypecanbewritten αα/αα.Asinthecaseof β thalassemia,therearetwomajorvarietiesof α thalassemia, α+ and α0 thalassemia.In α+ thalassemiaoneofthelinked α globingenesislost,eitherbydeletion( )ormutation(T);the heterozygousgenotypecanbewritten–α/αα or αT α/αα In α0 thalassemiathelossofboth α globingenesnearly alwaysresultsfromadeletion;theheterozygousgenotypeis thereforewritten /αα.Inpopulationswherespecificdeletionsareparticularlycommon,SoutheastAsia(SEA)orthe Mediterraneanregion(MED),itisusefultoaddtheappropriatesuperscriptasfollows:––SEA /αα or––MED /αα.Itfollows thatwhenwespeakofan“α thalassemiagene,”whatweare reallyreferringtoisahaplotype;thatis,thestateandfunction ofbothofthelinked α globingenes.
α0 thalassemia
Threemainmolecularpathologies,allinvolvingdeletions, havebeenfoundtounderliethe α0 thalassemiaphenotype. Themajorityofcasesresultfromdeletionsthatremoveboth α globingenesandavaryinglengthofthe α globingene cluster(Figure1.6).Occasionally,however,the α globingene clusterisintact,butisinactivatedbyadeletionwhichinvolves themajorregulatoryregionHS40,40kbupstreamfromthe α globingenes,orthe α globingenesmaybelostaspartofa truncationofthetipoftheshortarmofchromosome16. Aswellasprovidinguswithanunderstandingofthe molecularbasisfor α0 thalassemia,detailedstudiesofthese deletionshaveyieldedmoregeneralinformationaboutthe mechanismsthatunderliethisformofmolecularpathology. Forexample,ithasbeenfoundthatthe5′ breakpointsofa numberofdeletionsofthe α globingeneclusterarelocated approximatelythesamedistanceapartandinthesameorder
alongthechromosomeastheirrespective3′ breakpoints; similarfindingshavebeenobservedindeletionsofthe β globingenecluster.Thesedeletionsseemtohaveresulted fromillegitimaterecombinationevents,whichhaveledtothe deletionofanintegralnumberofchromatinloopsasthey passthroughtheirnuclearattachmentpointsduringchromosomalreplication.AnotherlongdeletionhasbeencharacterizedinwhichanewpieceofDNAbridgesthetwobreakpointsinthe α globingenecluster.Theinsertedsequence originatesupstreamfromthe α globingenecluster,where normallyitisfoundinaninvertedorientationwithrespect tothatfoundbetweenthebreakpointsofthedeletion.Thus itappearstohavebeenincorporatedintothejunctionina waythatreflectsitscloseproximitytothedeletionbreakpoint regionduringreplication.Otherdeletionsseemtoberelated tothefamilyofAlu-repeats,simplerepeatsequencesthat arewidelydispersedthroughoutthegenome;onedeletion appearstohaveresultedfromasimplehomologousrecombinationbetweentworepeatsofthiskindthatareusually62kb apart.
Anumberofformsof α0 thalassemiaresultfromterminal truncationsoftheshortarmofchromosome16toasiteabout 50kbdistaltothe α globingenes.Thetelomericconsensus sequenceTTAGGGn hasbeenaddeddirectlytothesiteofthe break.Sincethesemutationsarestablyinherited,itappears thattelomericDNAaloneissufficienttostabilizetheendsof brokenchromosomes.
Quiterecently,twoothermolecularmechanismshave beenidentifiedasthecauseof α0 thalassemiawhich,though rare,mayhaveimportantimplicationsforanunderstandingofthemolecularpathologyofothergeneticdiseases.In onecase,adeletioninthe α globingeneclusterresulted inawidelyexpressedgene(LUC7L)becomingjuxtaposed toastructurallynormal α globingene.Althoughthelatter retainedallitsimportantregulatoryelements,itsexpression wassilenced.Itwasfoundinatransgenicmousemodelthat transcriptionofantisenseRNAmediatedthesilencingofthe
Fig.1.6Someofthedeletionsthatunderlie α0 and α+ thalassemia. Thecoloredrectangles beneaththe α globingeneclusterindicatethe lengthsofthedeletions.Theunshadedregions indicateuncertaintyabouttheprecisebreakpoints. Thethreesmalldeletionsatthebottomofthefigure representthecommon α+ thalassemiadeletions. HVR,highlyvariableregions.
α globingeneregion,afindingthatprovidesacompletely newmechanismforgeneticdisease.Inanothercaseof α0 thalassemia,inwhichnomoleculardefectscouldbedetected inthe α globingenecluster,again-of-functionregulatory polymorphismwasfoundintheregionbetweenthe α globin genesandtheirupstreamregulatoryelements.Thisalteration createsanewpromoter-likeelementthatinterfereswiththe normalactivationofalldownstream α-likeglobingenes. Inshort,detailedanalysisofthemolecularpathologyof the α0 thalassemiashasprovidedvaluableevidencenotonly abouthowlargedeletionsofgeneclustersarecaused,butalso aboutsomeofthecomplexmechanismsthatmayunderlie casesinwhichthe α geneclustersremainintact,butinwhich theirfunctioniscompletelysuppressed.
α+ thalassemia
Asmentionedearlier,the α+ thalassemiasresultfromthe inactivationofoneoftheduplicated α globingenes,byeither deletionorpointmutation.
�� + Thalassemiaduetogenedeletions.Therearetwocommonformsof α+ thalassemiathatareduetolossofone orotheroftheduplicated α globingenes, α3.7 and α4.2 , where3.7and4.2indicatethesizeofthedeletions.Theway inwhichthesedeletionshavebeengenerated,approximately 4kblong,wasprobablygeneratedbyanancientduplication event.Thehomologousregions,whicharedividedbysmall inserts,aredesignatedX,Y,andZ.TheduplicatedZboxes are3.7kbapartandtheXboxesare4.2kbapart.Theresult ofmisalignmentreflectstheunderlyingstructureofthe α globingenecomplex(Figure1.7).Each α genelieswithin aboundaryofhomologyandreciprocalcrossoverbetween thesesegmentsatmeiosis,achromosomeisproducedwith eitherasingle( α)ortriplicated(ααα) α globingene.As showninFigure1.7,ifacrossoveroccursbetweenhomologousZboxes3.7kbofDNAarelost,aneventwhichis describedasarightwarddeletion, α3.7 .Asimilarcrossover
