Characterization of the Chediak-Higashi syndrome gene in human and mouse

203 Pages·1998·7.2 MB·English

by  Tchernev, Velizar T.

CHARACTERIZATIONOFTHECHEDIAK-HIGASHISYNDROMEGENEIN
HUMANANDMOUSE
By
VELIZART.TCHERNEV
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1998
Copyright1998
by
VelizarT.Tchernev
Tomyparents
ACKNOWLEDGMENTS
Theauthorwouldliketothankhismentor,Dr.StephenF.Kingsmore,andthe
supervisorycommitteemembers,Dr.M.Wallace,Dr.E.Wakeland,Dr.E.SobelandDr.
M.Bubb,fortheirvaluableadviceandencouragement.
Theauthor'sappreciationisextendedtoallformerandpresentmembersofDr.
Kingsmore'slaboratory,Dr.MariaD.F.S.Barbosa,Dr.VishnuS.Mishra,JohnC.Detter,
Dr.ElizabethB.McMurtrie,QuanA.Nguyen,SandraM.Holt,JuanM.Teodoroand
AndreaHofig, as well asto Dr. KrishnanNandabalan and Dr. Madan Kumar from
CuraGenCorporation,whocontributedtothesuccessfulcompletionofthiswork.
TheauthorwishestoexpresshisgratitudetoMs.AnnaV.GueorguievaandMr.
NathanS.Collierfortheirkindnessandcontinuoussupport.
Finally,butmostimportantly,theauthorwouldliketothankhisparentsLilia
JelevaandTzvetanTchernevfortheirlovingcareandguidance,hisbelovedwifeRalitza
Gueorguievaforherinvaluable supportandinspiration, andhis fellow-players Ivajlo
Kortezov,RadaKortezova,LudwigLubih,AnastasAssenovandIsakBerahafortheir
truefriendshipandthought-provokingconversations.
IV
TABLEOFCONTENTS
page
ACKNOWLEDGMENTS iv
ABSTRACT vii
INTRODUCTION 1
MATERIALSANDMETHODS 22
ReverseTranscription-PolymeraseChainReaction(RT-PCR)
AmplificationofCHSPatientandBeigeMousecDNAs 22
TACloningofPCRProductsandPlasmidPurification 23
Single-StrandConformationPolymorphism(SSCP)Analysis 24
DNASequencingandSequenceAnalysis 24
Allele-SpecificOligonucleotideAnalysis 25
PCRAmplificationofMouseLystcDNAIsoforms 25
CloningofHumanZESTcDNAs 26
MolecularProbes 28
IsolationofLYST2cDNAClones 29
SouthernBlotAnalysisandAutoradiography 30
NorthernBlotAnalysis 30
SimpleSequenceLengthPolymorphism(SSLP)PCRAmplification 31
BackcrossMousePanelandGeneticMapping 31
CloningofLYSTandLYST2BaitcDNAFragmentsinYeastTwo-
HybridVectors 32
YeastTwo-HybridScreens 36
ConfirmationoftheSpecificityofProteinInteractionsbytheYeast
Two-HybridSystem 40
RESULTS 43
IdentificationofMutationsinPatientswithChediak-Higashi
SyndromeandinBeigeMice 43
IdentificationandCharacterizationofLystmRNAIsoforms 52
IdentificationandCharacterizationoiLyst2,aBrain-SpecificMember
oftheChediak-HigashiSyndromeGeneFamily 61
IdentificationandSequenceAnalysis 61
GeneticMapping 62
ExpressionAnalysis 67
IdentificationofProteinsthatInteractwiththeCHSProteinandwith
LYST2,UsingaYeastTwo-HybridApproach 71
IP-1(ESTcg50136.f6,APPENDIX,Sequence13) 76
IP-2(ESTAA010799,APPENDIX,Sequence14) 77
IP-3(ESTcg50136.cl0,APPENDIX,Sequence15) 77
IP-4(ESTcg50136.a7,APPENDIX,Sequenced) 78
IP-5(ESTcg50175.c7,APPENDIX,Sequence17) 79
IP-6(ESTcg50138.g5,APPENDIX,Sequence18) 80
IP-7(ESTcg50173.dl0,APPENDIX,Sequence19) 81
IP-8(ESTcg50175.h7,APPENDIX,Sequence20) 82
IP-9(ESTKIAA0192,APPENDIX,Sequence21) 83
IP-10(ESTcg50136.a5.b,APPENDIX,Sequence22) 83
IP-11(ESTcg51287.dl0,APPENDIX,Sequence23) 84
IP-12(ESTcg49432.h3.b,APPENDIX,Sequence24) 85
IP-13(ESTcg50175.cl1,APPENDIX,Sequence25) 86
DISCUSSION 88
IdentificationandCharacterizationofLystmRNAIsoforms 88
IdentificationofMutationsinPatientswithChediak-Higashi
SyndromeandinBeigeMice 92
IdentificationandCharacterizationofLystl,aBrain-SpecificMember
oftheChediak-HigashiSyndromeGeneFamily 94
IdentificationofProteinsthatInteractwiththeCHSProtein,Usinga
YeastTwo-HybridApproach 98
APPENDIX SEQUENCESOFMOLECULESINTERACTINGWITHLYST
ANDLYST2 123
BIBLIOGRAPHY 177
BIOGRAPHICALSKETCH 192
VI
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
CHARACTERIZATIONOFTHECHEDIAK-HIGASHISYNDROMEGENEIN
HUMANANDMOUSE
By
VelizarT.Tchernev
December1998
Chairman:StephenF.Kingsmore
MajorDepartment:Pathology,ImmunologyandLaboratoryMedicine
Chediak-Higashisyndrome(CHS)isanautosomalrecessive,immunedeficiency
disorderofhumanandmouse(beige,bg)thatischaracterizedbyabnormalintracellular
proteintransporttoandfromthelysosome/lateendosome.Recentreportshavedescribed
thepositionalcloningofhomologousgenes,LYSTandLyst,thataremutatedinhuman
CHSandinbgmice,respectively.However,sincetheencodedproteinswerenoveland
unlike any of the molecules previously implicated in vesicular trafficking, their
identificationdidnotproveimmediatelyhelpfulinestablishingtheprecisemechanism
wherebyCHSdysregulatesproteintransport.Therefore,themaingoalofthisdissertation
was to characterize the Chediak-Higashi gene and its products inmore detail using
functionalgenomicsapproaches.Wedemonstratedthateachofthepreviouslyreported
bggenesequencesarederivedfromasinglegenewithalternativelysplicedmRNAs.
Alternativesplicingresultsinbggeneisoforms(Lyst-landLyst-IT)thatcontaindifferent
vn
3' regions. Similarly to mouse, the human mRNA isoforms arise from incomplete
splicingandretentionofatranscribedintronthatencodestheC-terminusofthepredicted
LYSTprotein.Additionalsplicingcomplexityofsmallerisoformsexists.TheLyst-lll
isoform lacksexonsa and p\whileinLyst-YV, exons a, P andyare absent. Novel
mutationswereidentifiedwithinthecodingdomainofLYSTinseveralCHSpatientsand
bgalleles.Interestingly,allbgandLYSTmutationsidentifiedtodatearepredictedto
resultineithertruncatedorabsentproteins.Mutationandexpressionanalysessuggested
thatdefectsinthefull-lengthmRNAalonecanelicitChediak-Higashisyndromeandthat
expressionofthesmallerisoformalonecannotcompensateforlossofthelargestisoform.
Wehavealsoidentifiedanovelgeneinhuman(LYST2)andmouse(Lyst2)thatappears
tobearelativeoftheCHSgene,basedonsequencesimilarity,predictedproteinstructure
andonsimilartranscriptsize.ComparisonoftherelativeabundanceofLYST2andLYST
mRNAssuggeststhatLYST2isexpressedabundantlyonlyinbrainandthereforeitmay
representabrain-specificmemberoftheCHSgenefamily.Usingamodifiedyeasttwo-
hybridsystem,ahumancDNAlibrarywasscreenedwithbaitsfromthecodingdomains
ofZTSTandLYST2.Severalproteins,whichplayimportantrolesinproteintransportand
signaltransduction,suchas14-3-3,caseinkinaseII,calmodulinandHrs,werefoundto
interactwithLYSTand/orLYST2.Manyoftheinteractingproteinscouldbelinkedina
commonpathwaythatregulatesvesiculartraffickinganddegranulation.
vin
INTRODUCTION
Chediak-Higashisyndrome(CHS)isaprimaryimmunodeficiency,characterized
bysevererecurrentinfections(Blume&Wolff,1972),partialalbinism(Windhorstetal,
1968),nervoussystemabnormalities(Misraetal, 1991),highincidenceofmalignancy
(Hayakawaetal,1986)andpredispositiontobleeding(Meyersetal,1974).Eighty-five
percentofpatientsundergoanacceleratedlymphoma-likephase,characterizedbyfever,
hepatosplenomegaly, lymphadenopathy, pancytopenia, coagulopathy and widespread
lymphohistiocytic organ infiltrates. Thiscomplication is fatal inthe absence ofbone
marrowtransplantation.
Thefirst3casesofChediak-HigashisyndromewerereportedbyBeguez-Cesarin
1943,thenonecasebySteinbrinkin 1948,andonecasebyHigashiin1954.Chediak
publishedcertainaspectsofBeguez-Cesar'scasesin1952,andSatoin1955recognized
thesimilaritiesbetweenChediak'sandHigashi'sdata,inventingtheeponym"Chediak-
Higashisyndrome."Exceptfortheoccasionalusageofsinglenamesorcombinationsof
the above authors' names, the disease has been most commonly referred to as the
Chediak-Higashisyndrome.
Approximately150casesofCHShavebeenreportedintheliterature.Malesand
femalesareaffectedequally. ThemeanageofdeathofCHSpatientsis6years. The
Chediak-Higashisyndromeoccurswithalowfrequencythroughouttheworld.Europe,
1
AmericaandJapanaretheareasfromwhichthemajorityoftheknowncaseshavebeen
described.Whilemostofthereportsdealwithisolatedcases,auniqueclusterofChediak-
Higashi syndrome patients has also been published (Ramirez-Duque et al, 1982).
Fourteencases ofCHS were found in 12 families living inadefined and relatively
isolatedgeographicalarea(Pregonero)intheVenezuelanAndes.Thepatientswerepre-
schoolchildrenexceptone11-year-oldfemale.Sixofthepatientsweremale.Allshowed
the sametypical clinical characteristics ofthe syndrome. SinceCHS isanautosomal
recessive disease (Sato, 1955), the existence ofthis high-frequency cluster ofCHS
patients inarestricted regioncould be explained by afoundereffect in an isolated
populationwithinbreeding.
Thepresenceofgiant,perinuclearvesicleswithproteinmarkerscharacteristicof
late endosomes andmature lysosomes is pathognomonic for CHS (Burkhardt etal,
1993). The organelles are abnormal both in size (enlarged) and in cytoplasmic
distribution(perinuclear).Whilethelateendosomal/lysosomalcompartmentisuniformly
affectedinCHS,thehistologicaldesignationofaffectedorganellesvariesbycelltype:
melanosomesareaffectedinCHSmelanocytes,whilesecretorygranulesareaffectedin
CHSneutrophils,eosinophils,mastcells,cytolyticT-cells,proximalrenaltubules,and
lysosomesareaffectedinmonocytesandfibroblasts.Thegiantorganellesarethoughtto
resultfromdysregulatedfusionofprimaryendosomesandlysosomes(Burkhardtetal.,
1993; Willingham et al, 1981). The morphological abnormalities ofbg and CHS
lysosomesareaccompaniedbycompartmentalmis-sortingofproteinssuchaselastase,(3-
glucuronidase,andcathepsinG(Holcombeetal.,1994).