revbrashematolhemoter.2017;39(1):63–65
w w w . r b h h . o r g
Revista
Brasileira
de
Hematologia
e
Hemoterapia
Brazilian
Journal
of
Hematology
and
Hemotherapy
Case
Report
First
observation
of
Hb
Ullevaal
[

78(EF2)
Leu>Val]
in
Turkey
F.
Sinem
Hocaoglu-Emre
a,∗,
Guven
Yenmis
b,
Cengiz
Yakicier
caDuzenLaboratoriesGroup,Istanbul,Turkey
bIstanbulUniversity,CerrahpasaMedicalFaculty,Istanbul,Turkey
cAcibademUniversity,Istanbul,Turkey
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t
i
c
l
e
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n
f
o
Articlehistory:
Received26August2016 Accepted30September2016 Availableonline10November2016
Introduction
Sofar,morethan800mutationsinvolvingthebeta-globingene havebeencharacterizedworldwide.Mostofthesevariantsare alsocarriedbypatientswithTurkishorigin,sinceTurkeyisone ofthehotspotregionsformutationsoftheglobingenes.1
Inthe present study,we reporta casewithan unusual high-performanceliquidchromatography(HPLC)patternwho turnedouttobeacarrierofthe78(EF2)Leu>Val;HBB:c.235
C>Gmutation.Thismutationispreviouslyunreportedin
Turk-ishindividuals.
Case
report
Thepatientattendedourlaboratoryforhematologicalexams duetopersistentanemiaandhemoglobinopathyHPLC anal-ysis was ordered as hemoglobinopathies are common in patientsofTurkishdescent.
Theproband wasa70-year-oldfemale fromthe eastern BlackSearegion.
∗ Correspondingauthorat:DuzenLaboratuvari,MecidiyekoyMah,CemalSahirSok,No:14,Sisli,Istanbul,Turkey.
E-mailaddress:sinememre@duzen.com.tr(F.S.Hocaoglu-Emre).
Thecompletebloodcountoftheprobandrevealedared bloodcell(RBC)countof3.36×103/L,hemoglobin(Hb)levelof
8.7g/dL,meancorpuscularvolume(MCV)of81.1fLandmean corpuscularHb(MCH)of25.8pg(Table1).
HPLCanalysiswasperformedusinganin-housemethod employinganultra-speedHPLCsystemwitha415nm UV/VIS-Detector(ShimadzuCorp.,Japan).ThemethodtoobtainaHb chromatogramwasmodifiedfromOuetal.2Theefficiencyof
thesystemiscontrolledpriortoeveryanalysisusinga ready-to-usetestsolutioncontainingtheHbvariantsHbA1c,HbF,Hb A,HbE,HbA2,HbD,HbSandHbC,andcontrolhemolysatesat twolevelsofconcentration,eachcontainingHbF,HbA,HbA2 andHbS(RECIPEChemicals+InstrumentsGmbH,Germany). In the HPLCanalysis, the Hb A level was38.8%, witha slightlyincreasedHbA2level(3.99%).NoHbFpeakwasseen inthefirstandfollowinganalyses.Besidesthis,anunknown 57.3%peakwasidentifiedintheHbEwindowadjacenttothe Hb A peak, indicating apossible heterozygoushemoglobin mutation.Thesamplewasrerunandexhibitedanidentical pattern(Figure1).
Inordertohaveadetailedanalysisofthesuspected muta-tion,thebetaglobingenewasamplifiedbypolymerasechain
http://dx.doi.org/10.1016/j.bjhh.2016.09.013
64
revbrashematolhemoter.2017;39(1):63–65Table1–Thehematologicalandmoleculardataofthe
patient.
Parameter Proband Referencerange
Age(years)–gender 70,F –
Hb(g/dL) 8.7 13.0–17.0
RBC(×103/L) 3.36 4.0–5.2
MCV(fL) 81.13 80–92
MCH(pg) 25.84 27–31
RDW-CV(%) 11.88 11.5–14.5
HbA(%):retention time/min
35.01:2.618 94–99
HbA2(%):retention time/min
3.99:3.141 0–3.90
Unknownpeak(%): retentiontime/min
57.31:2.701 –
SerumLDHlevel(U/L) 257 90–241
Serumironlevel(g/dL) 32 40–170
-Globingenotype c.235C>G/A A/A
Hb:Hemoglobin;RBC:redbloodcellcount;MCV:meancorpuscular volume;MCH:meancorpuscularhemoglobin;RDW:redbloodcell distributionwidth;LDH:lactatedehydrogenase.
Referencerangesusedarebasedontheequipmentmanufacturers’ establishedvaluesforthegenderandagegroupoftheproband.
reaction(PCR) andthe PCRproductwasanalyzedbydirect nucleotidesequencingusingaBeckmanCoulterCEQ2000XL DNAsequencer.
DNAsequencingoftheentirebetaglobingeneandthe pro-moterregionrevealedtheprobandwasheterozygousforthe
78(EF2)Leu>Val;HBB:c.235C>Gmutation,namedHbUllevaal
asdescribedbyGrimholtetal.3(Figure2).
Thisvarianthaspreviouslybeenreportedinonlyone per-son, a 61-year-old female Norway resident from Bosnia, a communitywithculturalandeconomicalaspectssimilarto TurkeysincetheOttomanEmpire,suggestingahistoricalpath forthemutation.Althoughnoabnormalpatternwasobserved byHPLCanalysisandthemutationwasdetectedincidentally whenthepatientwasreferredforthemeasurementofHbA1c
mV75
Detector A415nm
50
25
0
0 1 2
1463
2618 2701
3141
3 4 5 6
min Detector A415 nm
Area Variant Conc.%
Peak No Retention Time (min)
1 1.463 18 023 HbA1c
HbA
3681
35 015
57 311
3993
100 000 HbA2
171 448
280 623
19 553 2.618
3.141 2.701 2
3
4
Total
Figure1–Patternofthepatientwith78(EF2)Leu>Val;
HBB:c.235C>Gmutationusinganin-house
high-performanceliquidchromatography(HPLC)system.In
ourmethod,areferenceHPLCpatterncomprisesHbA1c,Hb
F,HbAandHbA2chainsrespectivelywiththeirorderof
appearanceonthechromatogram.However,anabnormal
variantpeak(indicatedwithanarrow)wasobservedinthe
probandonthe2.7thminuteofthemigrationthroughthe
columnwithaconcentrationof57.3%.Additionally,HbA2
level(retentiontime:3.141min)oftheprobandwasslightly increased(3.99%comparedtothereferencerangeof 0–3.90%).
bycationexchangeHPLC,thevariantHbpeakresidedbetween HbAandHbA2ontheHPLCchromatograminthefirstreport definingthissubstitution.Additionally,moderateanemiawith decreasedRBC,Hb,MCV,MCHandironlevelsandaslightly elevatedLDHconcentrationwaspresentintheproband.
revbrashematolhemoter.2017;39(1):63–65
65
Unfortunately,wewerenotabletoanalyzeother family membersgenetically. However,sequence analysisinvolving the alpha globingene and beta-globin gene promoterregion resultedinnovariations.
As Leucine and Valine are both basic aminoacids and this substitution does not change the net charge of the beta-globinmolecule,acomputerizedanalysisusingthe Gar-nierOsguthorpeRobson(GOR)methodforproteinstructure predictionsoftwarerevealedanalteredbetahelixstructure. Theloopanglecorrespondingtothesubstitutedaminoacid regionwasnarrowerassumingaconformationalchange in theprotein.
Thebetaglobingeneclusterhaplotypesofthismutation arelinkedtohaplotype1(+−−−−++)inourcase.4
Inconclusion,asthefirstreportofthedescribedmutation suggeststhereshouldbenoanemiaintheproband,the ane-miaseeninthiscasemightbearesultofirondeficiencyor otherconditions.Thisveryfirstreportofthe78(EF2)Leu>Val;
HBB:c.235C>GmutationinaTurkish individualhighlights
thegreatdiversityofthebetaglobingenepresentinthe Turk-ishpopulationandcontributesknowledgeofthemutational relationshipina‘benchtop archeological’mannerbetween differentcommunitiesduetohistoricalconnections.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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1.PatrinosGP,GiardineB,RiemerC,MillerW,ChuiDH,Anagnou NP,etal.ImprovementsintheHbVardatabaseofhuman hemoglobinvariantsandthalassemiamutationsfor
populationandsequencevariationstudies.NucleicAcidsRes. 2004;32:D537–41(databaseissue).
2.OuCN,RognerudCL.Rapidanalysisofhemoglobinvariantsby cation-exchangeHPLC.ClinChem.1993;39(5):820–4.
3.GrimholtRM,SudmannÅA,PiehlerAP,UrdalP,KlingenbergO. HbUllevaal[78(EF2)Leu→Val;HBB:c.235C>G],anew hemoglobinvariantinterferingwithHbA1cmeasurement
usingacationexchangehighperformanceliquid chromatographymethod.Hemoglobin.2014;38(2):130–2. 4.ÖztürkO,AtalayA,KöselerA,ÖzkanA,KoyuncuH,BayramJ,
