Rev. Bras. Hematol. Hemoter. vol.38 número3

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w w w . r b h h . o r g

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Original

article

Clinical,

hematological

and

genetic

data

of

a

cohort

of

children

with

hemoglobin

SD

Paulo

do

Val

Rezende

a,∗

,

Kenia

da

Silva

Costa

a

,

Jose

Carlos

Domingues

Junior

a

,

Paula

Barezani

Silveira

a

_,

_André

_Rolim

_Belisário

a

_,

_Celia

_Maria

_Silva

a

_,

Marcos

Borato

Viana

b

a_Fundac¸ão_Hemominas,_Belo_Horizonte,_MG,_Brazil

b_Universidade_Federal_de_Minas_Gerais_(UFMG),_Belo_Horizonte,_MG,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received9April2016

Accepted2May2016

Availableonline21May2016

Keywords:

Sicklecelldisease

HemoglobinS/D-Punjab

HemoglobinS-KorleBu

Children Haplotypes

a

b

s

t

r

a

c

t

Introduction:ThehemoglobinFSDisveryuncommoninnewbornscreeningprogramsfor

sicklecelldisease.IntheprogramofMinasGerais,Brazil,theclinicalcourseofchildren

withhemoglobinSDwasobservedtobeheterogeneous.Theobjectiveofthisstudywas

toestimatetheincidence(1999–2012)andtodescribethenaturalhistoryofacohortof

newbornswithhemoglobinSD.

Methods:Isoelectricfocusingwastheprimarymethodusedinnewbornscreening.

Poly-merasechainreaction-restrictionfragmentlength polymorphismandgene sequencing

wereusedtoidentifymutantallelesandforhaplotyping.Gap-polymerasechainreaction

wasusedtodetectalpha-thalassemia.

Results:ElevencasesofhemoglobinS/D-PunjabandeightofHbS-KorleBuweredetected.

OthervariantswithhemoglobinDmobilitywerenotidentified.AllhemoglobinD-Punjab

andhemoglobinKorleBualleleswereassociatedwithhaplotypeI.Amongthechildrenwith

hemoglobinS/D-Punjab,therewerefourwiththe␤SCARhaplotype,sixwiththeBenin

haplotype,andoneatypical.ResultsoflaboratorytestsforhemoglobinS/D-Punjaband

hemoglobinS-KorleBuwere:hemoglobin8.0and12.3g/dL(p-value<0.001),leukocytecount

13.9×109_/L_and_10.5_×₁₀9_/L_(p_-value₌_0.003),_{reticulocytes}_7.5%_and_1.0%₍_p_-value_<0.001),

hemoglobinFconcentration16.1%and6.9%(p-value=0.001)andoxygensaturation91.9%

and97%(p-value=0.002),respectively.OnlyhemoglobinS/D-Punjabchildrenhadacutepain

crisesandneededbloodtransfusionsorhydroxyurea.ThosewiththeBenin␤Shaplotypehad

highertotalhemoglobinandhemoglobinFconcentrationscomparedtotheCARhaplotype.

TranscranialDopplerwasnormalinallchildren.

Conclusion:TheclinicalcourseandbloodcellcountsofchildrenwithhemoglobinS/D-Punjab

wereverysimilartothoseofhemoglobinSSchildren.Incontrast,childrenwithhemoglobin

S-KorleBuhadclinicalcourseandbloodcellcountslikechildrenwiththesicklecelltrait.

byElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

∗ _{Corresponding}_author_at_:_Rua_Frei_Gonzaga,_301,_30315–170_Belo_Horizonte,_MG,_Brazil.

E-mailaddress:[email protected](P.d.V.Rezende).

http://dx.doi.org/10.1016/j.bjhh.2016.05.002

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Introduction

Sickle cell disease (SCD)is apublic healthproblem

world-wide. The hemoglobin (Hb) SD subtype seems to be very

rare. ItincludestheHb S/D-Punjabvariantthat apparently

isassociatedwithamoresevereclinical course,and other

HbS-non-Punjabvariants,withlimitedinformationregarding

laboratoryandclinicaldata.1–3

TheHbD-PunjabvariantisthemostcommonD-subtype

describedintheliteratureworldwide.Itwasfirstdescribedin

1951byItano.4_It_results_from_the_replacement_of_the_amino

acidglutamatewithglutamine atposition121ofthe

beta-globinchain[beta121(GH4)Glu>Gln;HBB:c.364G>C].

AnothervariantwithinthewindowofHbDusing

isoelec-tricfocalization(IEF),butnotusinghigh-performanceliquid

chromatography(HPLC),isHb KorleBu,whichresultsfrom

thereplacementoftheaminoacidaspartatewithasparagine

atposition72ofthebeta-globinchain[beta73(E17)Asp>Asn;

HBB:c.220G>A].ItoriginatesfromthewesternregionofAfrica,

andits dissemination toAmericais probablylinkedtothe

slavetradeinthe17thto19thcenturies.5

TheclinicalcourseofpatientswithHbSDdiseaseseems

tobeheterogeneous, depending onthe Hb D variant. This

suggeststhatthe sicklingprocessislikelytheresultofthe

interactionbetweentheintracellularHbSandHbDvariants.

Thisinteractionmaybestrengthenedorweakened,

depend-ingontheHbvariantco-inheritedwiththe␤Smutation.6–14

Children with Hb S/D-Punjab disease seem to present a

clinical course similar to those with homozygous Hb SS

disease.8,10,11,15,16

Thus,patientswithHbS/D-Punjabdiseaseshouldreceive

the same treatment protocol as those with Hb SS disease

becausetheymay alsoexperience potentiallyfatal

compli-cationsduringtheirlives.10,11,17 _The_use _of_hydroxyurea _in

childrenwithHbSDdiseasehasbeenrestrictedtoisolated

cases.11,18,19

Theobjectiveofthisstudywastoestimatetheincidence

andtodescribethenaturalhistoryofnewbornswiththeHb

SDpatternbyIEF,screenedaspartoftheNeonatalScreening

ProgramintheBrazilianstateofMinasGerais(PTN-MG).

Addi-tionally,beta-globinclusterhaplotypesforthe␤S,␤D-Punjaband

␤KorleBumutationsweredeterminedinordertotrytotracethe

originoftherespectivemutations.

Methods

This descriptive study is based on a retrospective cohort.

Archived medical records from the Fundac¸ão Hemominas

(GovernmentBloodCenter)andthePTN-MGdatabankwere

used.IEFofdriedbloodspotsamples(NeonatalHemoglobin

ResolveScreenKit,PerkinElmerLifeandAnalyticalSciences,

Finland)hasbeentheprimarymethodofnewbornscreening

atPTN-MG.Allele-specificpolymerasechainreaction(PCR)for

␤A,␤S,␤C,and␤D-Punjaballeleswasintroducedintheblood

bankprotocolasaconfirmatorytestin2010.

Thepopulationinitiallycomprised21childrenwithHbFSD

atbirth.HbFSDmeansthatthechildrenwerebornwiththree

Hbfractions:Hb F(themajorfractionatbirththatsteadily

Molecular Markers

600 bp

A

B

500 bp 400 bp

300 bp

200 bp

C C T T T A G TG/A A T G G C C T G

1 2 3 4 5

572 bp

300 bp

272 bp

Figure1–MoleculardetectionofmutationsinHbD-Punjab

andHbKorleBu.(A)Polymerasechainreaction-restriction

fragmentlengthpolymorphismwithEcoRI_in_five_children

withHbD-Punjab.Patients1,2,and4hadwildalleles(two

bandsof300and272basepairs)andPatients3and5had

anadditionalbandof572basepairsthatindicatesa

heterozygousmutationinthecodon121ofHBB_(Hb

D-Punjab);(B)electropherogramofachildwithHbKorleBu.

Thearrowpointstotheheterozygousmutation

HBB:c.220G>A(GAT>AAT;Asp>Asn)detectedthroughgene sequencing.

decreasesoverthefirstyearoflife),HbS,andanHbwitha

zoneDmobilityinelectrophoresis.Theywerebornbetween

January1,1999andDecember 31,2012;atotalof3,590,315

childrenwerescreenedinthis period.Thepatients’clinical

andlaboratorydatauptoDecember31,2014werereviewedso

thatallchildren,excepttwo,hadbeenfollowedupforatleast

twoyears.

Twopatientswereexcludedfromtheclinicalandmolecular

analyses because ethylenediaminetetraacetic acid

(EDTA)-anticoagulated blood samples had not been obtained for

moleculartests:onechildmovedabroadandanothermoved

outofthestate.Contactwiththefamilieshasfailedthusfar.

ThesechildrenwereusedonlytocalculatetheincidenceofHb

SDinthecohort.

Fragments of the beta-globin gene (HBB) containing

exon 3 (forward primer 5′_{-TCATGCCTCTTTGCACCATTC-3}′_;

reverseprimer5′_{-CACTGACCTCCCACATTCCC-3}′₎_were

ampli-fied using PCR, and polymerase chain reaction-restriction

fragment length polymorphism (PCR-RFLP) was conducted

withtheEcoRIenzymetodetecttheD-Punjaballele(Figure1A).

Ifthereactionwasnegative,thethreeexonsoftheHBBgene

weresequencedtoidentifythemutationunderlyingtheother

Hbvariants(primercompositionavailableonrequest).DNA

sequencing was done in an ABI 3130 capillary sequencer

(Applied Biosystems, FosterCity, CA,USA). Figure 1B

illus-tratesthegenesequencingoftheregioninwhichtheHBB:

c.220G>AmutationunderlyingHbKorleBuislocated.

Detec-tionofsevenmorecommonHBAdeletionswascarriedoutby

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The␤-globingeneclusterhaplotypingwascarriedoutby

PCR-RFLPofsix restriction sites: HindIIIin the IVS-IIof G_␥

(rs113425530),HindIII inthe IVS-IIofA_␥_{(rs28440105),} _HincII

in␺␤(rs10128556), HincII3′ _to_␺␤_(without _rs),_HinfI ₅′ _to_␤

(rs16911905),andHinfI3′_to_␤_{(rs10837631).}_{Additionally,}_HincII

5′_to

␧(rs3834466)andAvaIIatIVS-II-16of␤(rs10768683)were

determinedbygenesequencing.Restrictionenzymesusedfor

thehaplotype analyseswere purchasedfrom New England

Biolabs,Inc.(Beverly,MA,USA).Classificationofhaplotypes

wasbasedonOrkinetal.21_and_Nagel_et_al._for_the_␤S_gene.22

Theassignmentofspecifichaplotypesinheterozygousstates

shouldbeinterpretedwithcautioniffamilystudiesorallele

cloning are notperformed. Inthe present study,the

inter-pretationoftheresultswasfacilitatedbyourpreviousreport

showingthatin206HbSSchildrenfromMinasGerais,98.5%

ofthe␤SchromosomeswereofthetypesCARorBen.23

ACoulterT-890hematologycounterwasusedtoperform

all blood cell counts. Reticulocytes were counted in blood

smears stained with brilliant cresyl blue. All

hematologi-calvaluesweretranscribedfromthemedicalrecordsinthe

absence ofacuteclinical manifestationsand atleast three

monthsaftertheuse ofbloodproducts.Themathematical

averageofeachitemwasconsideredasthebaselinevaluefor

eachpatient.TherelativebaselineconcentrationofHbFwas

obtainedfromtheHbelectrophoresisresultsreportedineach

patient’smedicalrecord.Theresultsofelectrophoresis

test-ingperformedattheoldestagepossiblewithinthefollow-up

periodwere usedaslongasthesamplehadbeencollected

aftertwoyearsofage.

TranscranialDoppler(TCD)examinationswereperformed

in14childrenandinterpretedbyasingleexpertusinga

Nico-let equipment (modelEME TC 2000, Nicolet, Madison,WI,

USA).High-risk TCD wasdefinedasatime-averaged mean

ofthemaximumvelocity(TAMMX)≥200cm/sintheinternal

carotidormiddlecerebralarteryasoriginallydefinedbystroke

preventioninsicklecell anemia(STOP)investigators.24 _The

examinationcouldnotbeperformedinfivepatients: three

examinationswereimpossiblebecauseofthelackof

cooper-ationonthepartofthechildrenandtwowerenotperformed

becausethechildrenfailedtoattendtheexamination.

Statisticalanalyseswere performedusing theStatistical

PackagefortheSocialSciencesprogram(SPSS),version20.0.

Quantitativeresultsareexpressedastheaverage±standard

deviationorasthemedianandrangewhendistributionwas

non-Gaussian.Prevalencewasexpressedasapercentageand

a95% confidenceinterval(CI)wasapplied.Unpaired ttests

wereusedtocomparemeanvaluesbetweenHbS/D-Punjab

andHbS-KorleBugroups.Testresultswereconsidered

signif-icantwhentheprobabilityofalphaerrorwas≤0.05.

ThestudywasapprovedbytheEthicsResearchCommittee

attheinvolvedinstitutions(caseNo.13327713.5.0000.5149).It

wasconductedinaccordancewiththeHelsinkiDeclarationas

revisedin2008.Patientsand/orguardianswereaskedtosign

theinformedconsentform.

Results

The incidence of Hb FSD at birth was 1:171,000 (95% CI:

1:120,000–1:299,000). Molecular analyses were applied to

samplesfrom19outofthe21childrenbecauseaspreviously

mentioned,samplecollectionfromtwochildrenwasnot

pos-sible.Theagesrangedfrom2.8to16.2years,withamedian

of8.9years.Thirteenchildrenweremale(68.4%)andsixwere

female(31.6%).

Outofthe19children,11children(tenfamilies)were

diag-nosedwithHbS/D-Punjabdiseaseandeightwerediagnosed

withHbS-KorleBu.Noothervariantswerefound.Gender

dis-tributionintheHbS/D-Punjabgroupwasfourfemales(36.4%)

andsevenmales(63.6%)andgenderdistributionintheHb

S-KorleBugroupwastwofemales(25%)andsixmales(75%).The

medianagewas11years(range:2.8–16.2)intheHbS/D-Punjab

groupand7.7years(range:3–13.7)intheHbS-KorleBugroup.

AllchildrenoftheHbS/D-Punjabgroupwerefoundtohave

baseline Hblevels below10g/dL(average: 8.0g/dL) and the

reticulocytecountsvaried.Thebaselinevaluesofthe

hema-tologic testsandgeneticresultsforeach childarereported

inTable1.Figure2comparesthemainresultsfoundinboth

groups. TheHbS/D-Punjabgroupwas foundtohave lower

average baseline Hb values and higher reticulocyte counts

thantheHbS-KorleBugroup(p-value<0.001forboth

com-parisons). Relative Hb Fconcentrations were higher in the

children ofthe HbS/D-Punjab group(p-value=0.001). Total

leukocyteandplateletcountswerealsohigherintheHb

S/D-Punjabgroup(p-value=0.003andp-value=0.06,respectively).

MeanratiosbetweenHbSandHbDconcentrationswere1.06

and 1.14 in the Hb S/D-Punjab and Hb S-KorleBu groups,

respectively(p-value=0.61).

The coinheritance of alpha-thalassemia (␣␣/−␣3.7) was

detected in four children (two with Hb S/D-Punjab and

two with Hb S-Korle Bu). Mean corpuscular volume (MCV)

and mean corpuscular Hb (MCH) were significantly lower

in children who co-inherited the alpha-thalassemia

dele-tion(p-value=0.008andp-value=0.03,respectively).TheHb

S/D-Punjabgroupwasanalyzedseparately:theMCVsofthe

patientswithandwithout-alphathalassemiaaveraged76.6fL

and85.9fL,respectively(p-value=0.001),whiletheMCHs

aver-aged24.2pgand29.1pg,respectively(p-value=0.001).

Intermsoftheassociatedclinicalfindings,two(18.2%)of

the childrenintheHb S/D-Punjabgrouphad acutesplenic

sequestrationcrises(ASSCs),andall11experiencedatleast

oneacutepainepisode.However,therewerenoASSCsinthe

HbS-KorleBugroup,andthreepatients(37.5%)hadatleast

oneacutepainepisodereportedassuchinthepatients’

med-icalrecords.NoneofthechildrenintheHbS-KorleBugroup

receivedbloodtransfusions;however,seven(63.6%)children

intheHbS/D-Punjabgroupreceivedtransfusions.

Nochildhadanovertstroke.MeanDopplerTAMMXvalues

forHbS/D-PunjabandHbS-KorleBuchildrenwere131.1cm/s

[standarderrorofthemean(SEM):6.7] and89.2cm/s(SEM:

6.5),respectively(p-value=0.001).Allchildrenwereclassified

asbeingatlowriskforstrokes.

BaselineoxygensaturationwaslowerintheHbS/D-Punjab

groupandsignificantlydifferedfromtheHbS-KorleBugroup.

Averagevaluesinthetwogroupswere91.9%and97%,

respec-tively (p-value=0.002). There were no deaths in the study

population.

Clinicaltreatmentwasalsoreviewed.Allpatientsreceived

aprescriptionforantimicrobialprophylaxis,dailyfolic acid

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Table1–Meanbaselinehematologicaldataandgeneticresultsof11childrenwithHbS/DPunjab_and_eight_children_with HbS-Korle-Bu.

Id/Gender Hemoglobin

(g/dL)

Leukocytes (×109_/L)

Platelets (×109_/L)

Reticulocytes (%)

HbF(%) HbS(%) HbD(%) ␤S

haplotype/␣−3.7 thalb

ChildrenwithHbS/DPunjab₍_n₌₁₁₎

1/F 7.2 13.9 347.7 4.0 13 48 37 CAR/−

2/M 7.9 16.1 431.8 14.7 13 45 39 ATP/−

3/F 6.8 13.7 368.0 8.7 14 38 44 BEN/−

4/M 6.8 18.0 552.0 7.5 4 44 37 CAR/−

5/F 8.3 15.2 300.2 9.5 22 41 35 BEN/−

6/Ma _8.6 _11.2 _335.0 _3.9 ₂₃ ₃₃ ₄₀ _BEN/+

7/F 9.5 12.6 353.8 3.2 16 47 39 BEN/−

8/Ma _8.9 _15.0 _454.0 _6.4 ₂₁ ₃₆ ₄₀ _BEN/₋

9/M 8.4 13.6 432.2 10.3 10 40 46 CAR/+

10/M 6.2 13.3 308.8 8.7 16 39 35 CAR/−

11/M 9.5 11.2 430.9 5.9 25 37 36 BEN/−

Mean 8.0 14.0 392.2 7.5 16.1 40.7 38.9

ChildrenwithHbS-Korle-Bu(n=8)

1/M 11.7 12.2 397.3 1.8 6 58 34 CAR/+

2/M 12.8 10.8 366.9 1.2 9 45 45 CAR/−

3/M 11.5 7.1 326.6 1.0 7 45 42 CAR/−

4/F 12.8 7.7 257.0 1.1 6 59 33 BEN/−

5/F 13.2 10.1 275.6 0.4 9 41 48 CAR/+

6/M 12.3 11.4 331.7 0.6 6 39 52 CAR/−

7/M 12.0 14.3 407.9 1.3 4 50 44 CAR/−

8/M 11.8 10.5 266.3 0.8 8 39 50 CAR/−

Mean 12.3 10.5 328.7 1.0 6.9 47.0 43.5

a _Brothers.

b _{Co-inheritance}₍₊_or₋₎_of_the_{alpha-thalassemia}_gene_−␣3.7₍_−␣3.7_/_␣␣_).

15.0

A

B

C

D

10.0

5.0

Mean hemoglobin concentr

ation (g/dL)

Mean f

etal hemoglobin, %

Mean leuk

ocyte count (x10E9/L)

Mean reticulocyte count, %

.0

25

20

10 15

0 5

20.0

15.0

5.0 10.0

0 15.0

10.0

5.0

.0 D-Punjab

P<.001

P=.001

P<.001

P=.003 Korle-Bu

D-Punjab Korle-Bu

Figure2–Meanbaselinehematologicaldataof11childrenwithHbS/D-PunjabandeightchildrenwithHbS-KorleBu.(A)

Totalhemoglobinconcentration(g/dL);(B)reticulocytes(%);(C)fetalhemoglobin(%);(D)leukocytecount(×109_/L)._The

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Table2–ClinicalandlaboratorialdatainthreechildrenwithHbSD-Punjabandresponsetohydroxyurea.

Id/gender Age(years)at startofHU

Clinical indication

forHU

Dose (mg/kg/day)

Duration (months)

Before hydroxyurea

After hydroxyurea

Decreased numberof clinicalevents TotalHb

(g/dL)/Hb F(%)

Ret(%) TotalHb (g/dL)/Hb F(%)

Ret(%)

11/M 14 Severe

vaso-occlusive painepisodes

20 8 9.5/26.7 6.1 11.1/34 0.4 Yes

9/M 6.6 Severe

27 76 8.5/9.0 8.4 10.2/17 3.4 Yes

4/Ma _0.8 _Anemia ₁₅ ₃₆

8 Severe

26 27 7.6/4.0 11.7 8.8/20 6.3 Yes

Id:identificationreferstonumbersinTable1;HU:hydroxyurea;Ret:reticulocytecount.

a _This_child_was_given_hydroxyurea_in_August₂₀₀₅_because_of_Hb_<_5.0_g/dL._Hydroxyurea_was_withdrawn_in_July₂₀₀₈_by_his_mother,_not_by_the

hematologist.ThedrugwasrestartedinSeptember2012,becauseofmultipleepisodesofvaso-occlusivepain.

childrenwithHbS/D-Punjabdiseasewerefoundtohaveused

hydroxyurea,asindicatedforrecurringepisodesofpain

cri-sis.AllchildrenpresentedincreasedtotalHbandHbFaswell

aslower reticulocyte countsand fewerpain episodes after

hydroxyureatherapy.Anotherpatientwhohadalsoreceived

anindicationfortheuseofhydroxyureabecauseofrecurring

paincrisesislikelytobeginusingthemedicationsoon.Table2

showsthedataontheuseofhydroxyurea.

HaplotypingofHbS/D-Punjabshowedthatallpatients

pre-sentedhaplotypeIfortheD-Punjaballele (+----+++,forthe

eightanalyzedpolymorphicsitesinthe5′_->3′ _direction)._In

respecttothe␤-globingenecluster,sixwereBenin,fourCAR,

andonehadanatypicalhaplotype(+----+++,e.g.,haplotypeI

inhomozygosis).Similarly,allHbS-KorleBuallelespresented

haplotypeI.Sevenhadthe␤SCARhaplotypeandonlyone,the

␤SBeninhaplotype.

Comparing hematological data between ␤S CAR and ␤S

Benin haplotypes withinthe Hb S/D-Punjab group of

chil-dren,Hbconcentration(7.2g/dLversus8.6g/dL,respectively)

andfetal Hb (10.8%versus20.2%,respectively)were

signif-icantly higher for the Benin haplotype (p-value=0.05 and

p-value=0.01,respectively).Nootherdata,includingDoppler

TAMMXvalues, oxygen saturation,andnumber of

transfu-sions, were significantlydifferent betweenCAR and Benin

groups.

Discussion

Two variants with the IEFprofile of Hb D were identified:

D-PunjabandKorleBu.Theclinicalandlaboratory

character-isticsofthetwogroupswereverydifferent.

Children with Hb S/D-Punjab disease presented many

different symptoms. This clinical courseis similar to that

observedinchildrenwithhomozygousHbSSdiseaseashas

alreadybeenreportedinotherstudies.10,11,16,17,25_Oberoi_et_al.

evaluatedten patientsagedbetween1and19 years.All of

them presentedwith moderate or severe anemia(average:

6.8g/dL)andatleastoneclinicalcomplicationrelatedtothe

disease,suchaspaincrisis,acutechestsyndrome,gallstones,

avascularnecrosisoffemoralhead,andrecurrentinfections.

Furthermore,eightpatientsrequiredanaverageofthreered

bloodcelltransfusionsduringtheirclinicalfollow-upperiod.11

In the present study, blood transfusions were required by

63.6%ofpatients.

Italiaetal.reportedon15patientswithagesbetween1and

34years.Theyclassifiedonlyonepatientasmild;twowere

classifiedashavingexperiencedmoderatecrises,and12were

foundtohaveseverehemolyticanemia;these12presented

recurrent pain crisesand requiredfrequent transfusions.10

El-Kalla & Mathews evaluated nine patients aged between

3and 10 yearsinthe UnitedArabEmirates. Theyreported

paincrisesofvaryingintensity, acutesplenicsequestration

crises,andrepeated infectionsinsevenpatients.Theother

twochildrenwereasymptomatic,althoughanemicandwith

increasedreticulocytecounts.17_The_largest_sample_size_in_the

literaturewaspublishedbyPateletal.Of42Indianpatients

with anaverage ageof22 years,25 were considered tobe

severe casesforpresentingthreeor moreacutepaincrises

and/ortheneedoftwoormorebloodtransfusionsintheyear

priortothestudy.25

ThegroupofchildrenwithHbS-KorleBudiseasedidnot

presentclinicalcomplications.Threechildrenpresentedwith

mildtomoderatediffuseabdominalpainthatwasconsidered

secondarytotheunderlyingdisease.However,apaincrisis

isfrequentlyinferredasthecauseofabdominalpainwhen

signsandsymptomsrelatedtootheretiologies(suchasfever,

vomiting,andbloodinthestools)areabsentinchildren.

Dur-ingthecrises,thesechildrenaregenerallyassumedtohaveHb

S/D-Punjabeveniftheyhavenotbeensubmittedtomolecular

testing.Paincrisesmaybedifficulttodistinguishfromother

equallyprevalentetiologiesinchildrensuchasconstipation

and functionalabdominalpain.26 _It_is_known _that

abdomi-nalpainaffects38%oftheschool-agedpediatricpopulation

weekly; noreliablebiologicalmarkersclearlydefine a

diag-nosisoffunctionalpain.27_In_addition,_few_studies_show_that

theclinicalcourseofHbS-KorleBudiseasecarriersmaybe

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ofpatientsandlengthoffollow-upprecludeasolid

conclu-sion.Someauthorshavecomparedthesepatientstosickle

celltraitcarriers.7_Therefore,_the_exact_definition_of_the_Hb_D

variantthatthepatienthasisimportantforthecorrect

dif-ferentialdiagnosisofabdominalpaininchildren.Italsoaids

indecreasingboththe unnecessarystigmaagainstchronic

diseasesandunnecessarytreatments,suchasthe

indiscrim-inate use of powerful anti-inflammatory medications and

painkillers.

Thereseems tobe noreports availableinthe literature

abouttranscranialDopplerexaminationsinchildrenwithHb

SDdisease.Inthepresentstudy,allchildrenwerefoundtobe

atlowriskforstrokes.Asexpected,themeanvalueofTAMMX

wassignificantlyhigherinHbS/D-PunjabthaninHbS-Korle

BuchildrenbecausetheHbconcentrationwas significantly

lowerintheformergroup.

HigherHbFbaselinepercentageswereobservedintheHb

S/D-Punjabgroup;therewasastatisticallysignificant

differ-encefrom thevaluesobservedintheHbS-KorleBugroup.

Theinfluenceofthispercentageondecreasingsymptomsin

childrenwithHbS/D-Punjabdisease remainsunclear. This

associationhasbeen described inpatientswithHb SS

dis-easeandhasbeenexplainedbythefactthatredbloodcells

withlargerquantitiesofHb Fpossess lowerlevelsofHbS;

therefore,theyhavealowerchanceofsickling,andasaresult,

alowerprobabilityofexperiencingclinicalmanifestations.28

Patel et al. found higher Hb F concentrations to be

asso-ciated with a lower frequency of acute pain crises in the

42patientswithHbS/D-Punjabdisease.25 _Meanwhile,_three

other studies failed to find this association;however, they

did include much smaller sample populations than Patel

etal.Theotherstudiesincludedfive,nine,and15patients,

respectively.9,10,17

ThreechildrenwiththeHbS/D-Punjabvarianttook

hydrox-yurea, and all childrenexperiencedsignificant clinical and

laboratoryimprovements.InchildrenwithHbSS,theuseof

hydroxyureahasbeendeterminedtobesafeandtoprovide

satisfactory results.29 _In _patients _with _Hb _SD _disease, _the

dataontheefficacyandsafetyofthismedicationarelimited

toisolated cases19 _and_to_a_few_studies_with_a_larger

num-berofcases.Out oftheten childrenincludedinthe study

byOberoietal.,fivereceivedhydroxyureatotreatrecurrent

painepisodesand/orsevereanemia.Theirfollow-upperiods

rangedfrom6to50months.Inallcases,therewasadecrease

inthe number ofpaincrisesand alack ofsignificantside

effects.11_The_largest_cohort_included₂₀_Indian_patients_aged

between1and 45years who tookhydroxyurea foratleast

twoyears.Decreasedfrequenciesofcrisesandinterruptions

intransfusionswererecordedforallpatients.Nosideeffects

wereobserved.18

HaplotypeIisthecommonesttypeofD-Punjaballele in

almost all ethnic populationsso far described.6,14,15,30 _The

exceptionisThailand,wherehaplotypeIIwasfoundinnine

patientsfromfivefamilies.31_So_it_is_impossible_to_trace_back

theoriginofthealleleinthepopulationofthisstudy.

Haplo-typesfortheHbKorleBuallelehavenotbeendescribedyet.

Becauseall childreninthepresent studyare alsotypeI, it

isimprobablethattheoriginoftheallelewillbedetermined

forsure.Itwouldbeinterestingtoknowwhetherthesame

haplotypeispresentinWesternAfricawherethemutation

seeminglyoriginated.5

Asfarasisknownfrominternationalreports,thepresent

studydemonstratedforthefirsttimethat,withintheHb

S/D-Punjabgroup,childrenwiththe␤SBeninhaplotypehadhigher

totalHbconcentrationsandhigherrelativeconcentrationsof

fetalHbthanchildrenwiththe␤SCARhaplotype.

Thelimitationsofthepresentstudyincludethefactthat

itwasaretrospectiveanalysisandalownumberofpatients

wereinvolved.Thesefactorslimitconclusionsregardingthe

incidenceofclinicalcomplicationsincasesofHbS/D-Punjab

andHbS-KorleBudiseaseaswellasareliableevaluationofthe

late-onseteffectsoftheuseofhydroxyurea.Furthermore,had

high performanceliquidchromatography(HPLC)been used

astheprimaryscreeningtestatPTN-MG,childrenwithHb

S-KorleBuwouldnotbeclassifiedashavingHbSD,butinstead

ashavinganunknownvariantwithintheHPLCwindowofHb

A2/HbE.

Inconclusion,thisisthefirststudyinBraziltoevaluate

childrenwithHbSDdiseasedetectedatbirthbyIEF.Itoffers

acleardescriptionoftwovariants:HbS/D-PunjabandHb

S-KorleBu.Earlynewbornscreeningandthesystematicgenetic

studyofHbDvariantsareusefulintreatingpatientsandin

informingthefamilymembers abouttheprognosisofeach

variant.ChildrenwithHb S/D-Punjabhaveaclinicalcourse

similartothosewithHbSSdisease.Thesubgroupwiththe␤S

CARhaplotypehavelowertotalHbconcentrationsandlower

relativeHbFconcentrationsthanthosewiththe␤SBenin

hap-lotype,butthelimitednumberofpatientsprecludesdefinitive

conclusions. In contrast,the clinical courseand laboratory

datainchildrenwithHb S-KorleBuappeartobesimilarto

thoseinchildrenwiththesicklecelltrait,butcautionshould

betakenwiththisstatement,consideringthelimitednumber

ofchildrenandshortfollow-upthusfarreported.

Funding

Fundac¸ãoHemominas,NewbornScreeningProgram

(Nupad-UFMG), Fundac¸ão de Amparo à Pesquisa de Minas Gerais

(FAPEMIG),andConselhoNacionaldeDesenvolvimento

Cien-tíficoeTecnológico(CNPq).

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Theauthorsacknowledgeallsubjectsand parentsfortheir

cooperationinthestudy.Theauthorsalsothankthe

finan-cial support of Fundac¸ão Hemominas, Newborn Screening

Program(Nupad-UFMG),Fundac¸ãodeAmparoàPesquisade

MinasGerais(FAPEMIG;grant#PPM-00780-15),andConselho

NacionaldeDesenvolvimentoCientíficoeTecnológico(CNPq;

(7)

r

e

f

e

r

e

n

c

e

s

1. SmithEW,ConleyCL.SicklecellhemoglobinDdisease.Ann InternMed.1959;50(1):94–105.

2. CaweinMJ,LappatEJ,BrangleRW,FarleyCH.HemoglobinS-D disease.AnnInternMed.1966;64(1):62–70.

3. TorresLS,OkumuraJV,SilvaDG,Bonini-DomingosCR. HemoglobinD-Punjab:origin,distributionandlaboratory diagnosis.RevBrasHematolHemoter.2015;37(2):120–6.

4. ItanoHA.Athirdabnormalhemoglobinassociatedwith hereditaryhemolyticanemia.ProcNatlAcadSciUSA. 1951;37(12):775–84.

5. HonigGR,SeelerRA,ShamsuddinM,VidaLN,MompointM, ValcourtE.HemoglobinKorleBuinaMexicanfamily. Hemoglobin.1983;7(2):185–9.

6. YavarianM,KarimiM,ParanF,NevenC,HarteveldCL, GiordanoPC.MulticentricoriginofHbD-Punjab [beta121(GH4)Glu→Gln,GAA>CAA].Hemoglobin. 2009;33(6):399–405.

7. AklPS,KutlarF,PatelN,SalisburyCL,LaneP,YoungAN. CompoundheterozygosityforhemoglobinS

[beta6(A3)Glu6Val]andhemoglobinKorleBu [beta73(E17)Asp73Asn].LabHematol.2009;15(3):20–4.

8. AdachiK,KimJ,BallasS,SurreyS,AsakuraT.Facilitationof HbSpolymerizationbythesubstitutionofGluforGlnatbeta 121.JBiolChem.1988;263(12):5607–10.

9. AdekileA,Mullah-AliA,AkarNA.Doeselevatedhemoglobin FmodulatethephenotypeinHbSD-LosAngeles?Acta Haematol.2010;123(3):135–9.

10.ItaliaK,UpadhyeD,DabkeP,KanganeH,ColacoS,SawantP, etal.ClinicalandhematologicalpresentationamongIndian patientswithcommonhemoglobinvariants.ClinChimActa. 2014;431:46–51.

11.OberoiS,DasR,TrehanA,AhluwaliaJ,BansalD,MalhotraP, etal.HbHbS/D-Punjab:clinicalandhematologicalprofileofa rarehemoglobinopathy.JPediatrHematolOncol.

2014;36(3):e140–4.

12.NagelRL,LinMJ,WitkowskaHE,FabryME,BestakM,Hirsch RE,etal.CompoundheterozygosityforhemoglobinCand KorleBu:moderatemicrocytichemolyticanemiaand accelerationofcrystalformation.Blood.1993;82(6):1907–12.

13.AdachiK,KimJ,KinneyTR,AsakuraT.Effectofthebeta73 aminoacidonthehydrophobicity,solubility,andthekinetics ofpolymerizationofdeoxyhemoglobinS.JBiolChem. 1987;262(22):10470–4.

14.PatelDK,MashonRS,PatelS,DashPM,DasBS.Beta-globin genehaplotypeslinkedwiththeHbD-Punjab

[beta121(GH4)Glu→Gln,GAA>CAA]mutationineasternIndia. Hemoglobin.2010;34(6):530–7.

15.RahimiZ,AkramipourR,KoraniS,NagelRL.HbD-Punjab [beta121(GH4)Glu→Gln]/beta0-thalassemia[IVSII.1(G→A)] intwocasesfromanIranianfamily:firstreport.AmJ Hematol.2006;81(4):302–3.

16.KelleherJFJr,ParkJO,KimHC,SchroederWA.

Life-threateningcomplicationsinachildwithhemoglobin SD-LosAngelesdisease.Hemoglobin.1984;8(3):203–13.

17.el-KallaS,MathewsAR.HbD-PunjabintheUnitedArab Emirates.Hemoglobin.1997;21(4):369–75.

18.PatelS,PurohitP,MashonRS,DehuryS,MeherS,SahooS, etal.Theeffectofhydroxyureaoncompoundheterozygotes forsicklecell-hemoglobinD-Punjab–asinglecentre experienceineasternIndia.PediatrBloodCancer. 2014;61(8):1341–6.

19.UddenMM,LoMN,SearsDA.Successfulhydroxyurea treatmentofapatientwithSDhemoglobinopathy.AmJ Hematol.1999;60(1):84–5.

20.TanAS,QuahTC,LowPS,ChongSS.Arapidandreliable 7-deletionmultiplexpolymerasechainreactionassayfor alpha-thalassemia.Blood.2001;98(1):250–1.

21.OrkinSH,KazazianHHJr,AntonarakisSE,GoffSC,BoehmCD, SextonJP,etal.Linkageofbeta-thalassaemiamutationsand beta-globingenepolymorphismswithDNApolymorphisms inhumanbeta-globingenecluster.Nature.

1982;296(5858):627–31.

22.NagelRL,FabryME,PagnierJ,ZohounI,WajcmanH,BaudinV, etal.Hematologicallyandgeneticallydistinctformsofsickle cellanemiainAfrica.TheSenegaltypeandtheBenintype.N EnglJMed.1985;312(14):880–4.

23.BelisárioAR,MartinsML,BritoAM,RodriguesCV,SilvaCM, VianaMB.␤-Globingeneclusterhaplotypesinacohortof221 childrenwithsicklecellanemiaorS␤◦_-thalassemia_and_their

associationwithclinicalandhematologicalfeatures.Acta Haematol.2010;124(3):162–70.

24.AdamsRJ,McKieVC,HsuL,FilesB,VichinskyE,PegelowC, etal.Preventionofafirststrokebytransfusionsinchildren withsicklecellanemiaandabnormalresultsontranscranial Dopplerultrasonography.NEnglJMed.1998;339(1):5–11.

25.PatelDK,PurohitP,DehuryS,DasP,DuttaA,MeherS,etal. Fetalhemoglobinandalphathalassemiamodulatethe phenotypicexpressionofHbHbS/D-Punjab.IntJLab Hematol.2014;36(4):444–50.

26.RhodesMM,BatesDG,AndrewsT,AdkinsL,ThorntonJ, DenhamJM.Abdominalpaininchildrenwithsicklecell disease.JClinGastroenterol.2014;48(2):99–105.

27.SapsM,SeshadriR,SztainbergM,SchafferG,MarshallBM,Di LorenzoC.Aprospectiveschool-basedstudyofabdominal painandothercommonsomaticcomplaintsinchildren.J Pediatr.2009;154(3):322–6.

28.SteinbergMH,RodgersGP.Pharmacologicmodulationoffetal hemoglobin.Medicine.2001;80(5):328–44.

29.ThornburgCD,FilesBA,LuoZ,MillerST,KalpatthiR,IyerR, etal.ImpactofhydroxyureaonclinicaleventsintheBABY HUGtrial.Blood.2012;120(22):4304–10.

30.AtalayEÖ,AtalayA,ÜstelE,YildizS,OztürkO,KöselerA, etal.GeneticoriginofHbD-LosAngeles[␤121(GH4)Glu→Gln, GAA→CAA]accordingtothe␤-globingenecluster

haplotypes.Hemoglobin.2007;31(3):387–91.

31.FucharoenS,ChangtrakunY,SurapotS,FucharoenG, SanchaisuriyaK.MolecularcharacterizationofHbD-Punjab [beta121(GH4)Glu→Gln]inThailand.Hemoglobin.