Molecular analysis and association with clinical and laboratory manifestations in children with sickle cell anemia

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

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

Original

article

Molecular

analysis

and

association

with

clinical

and

laboratory

manifestations

in

children

with

sickle

cell

anemia

Roberta

Faria

Camilo-Araújo

_,

_Olga

_Maria

_Silverio

_Amancio,

_Maria

_Stella

_Figueiredo,

Ana

Carolina

Cabanãs-Pedro,

Josefina

Aparecida

Pellegrini

Braga

UniversidadeFederaldeSãoPaulo(UNIFESP),SãoPaulo,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received1August2013

Accepted31March2014

Availableonline9July2014

Keywords:

Anemia,SickleCell

Alpha-thalassemia Beta-globins Haplotypes Child

a

b

s

t

r

a

c

t

Objectives:Toanalyzethefrequencyof␤S-globinhaplotypesandalpha-thalassemia,and

theirinfluenceonclinicalmanifestationsandthehematologicalprofileofchildrenwith

sicklecellanemia.

Method:Thefrequencyof␤S-globinhaplotypesandalpha-thalassemiaandanyassociation

withclinicalandlaboratorialmanifestationsweredeterminedin117sicklecellanemia

childrenaged3–71 months. The confirmation ofhemoglobin SS anddetermination of

thehaplotypeswereachievedbypolymerasechainreaction-restrictionfragmentlength

polymorphism,and alpha-thalassemia genotyping wasby multiplex polymerasechain

reaction(single-tubemultiplex-polymerasechainreaction).

Results:The genotype distribution of haplotypes was 43 (36.7%) Central African

Republic/Benin,41 (35.0%)Central AfricanRepublic/Central African Republic,20 (17.0%)

Rare/atypical,and13(11.1%)Benin/Benin.Thefrequencyofthe␣3.7deletionwas1.71%

as homozygous (−␣3.7/−␣3.7) and 11.9% as heterozygous (−␣3.7/␣␣). The only

signifi-cantassociationinrespecttohaplotypeswasrelated tothemeancorpuscularvolume.

The presence of alpha-thalassemia was significantly associated to decreases in mean

corpuscular volume, mean corpuscular hemoglobin and reticulocytecount and to an

increaseintheredbloodcellcount.Therewerenosignificantassociationsof␤S-globin

haplotypesandalpha-thalassemiawithclinicalmanifestations.

Conclusions:In the study population, the frequency of alpha-thalassemia was similar

topublished datainBrazilwiththeCentralAfricanRepublichaplotypebeingthemost

common,followedbytheBeninhaplotype.␤S-globinhaplotypesandinteractionbetween

alpha-thalassemiaandsicklecellanemiadidnotinfluencefetalhemoglobinconcentrations

orthenumberofclinicalmanifestations.

©2014Associac¸ãoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published

byElsevierEditoraLtda.Allrightsreserved.

∗ _{Correspondingauthorat:EscolaPaulistadeMedicina,UniversidadeFederaldeSãoPaulo(UNIFESP),RuaBotucatu,703,VilaClementino,}

04023-062SãoPaulo,SP,Brazil.

E-mailaddress:robertafc.dped@epm.br(R.F.Camilo-Araújo).

http://dx.doi.org/10.1016/j.bjhh.2014.06.002

1516-8484/©2014Associac¸ãoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.PublishedbyElsevierEditoraLtda.Allrights

Introduction

Sicklecellanemia(HbSS)ischaracterizedbyhomozygosity

forhemoglobinS(HbS),consequenttoapointmutationin

the6thcodonofthe␤-globingeneonchromosome11.1,2_Itis

themostprevalenthereditaryhematologicdiseaseinBrazil

withelevated morbidityand mortality.It isestimatedthat

3500childrenarebornwithHbSSannually,oneinevery1000

livebirths,whereasthefrequencyofheterozygotesinthe

gen-eralpopulationis2–8%.3,4_{Theclinicalstateofthesepatients}

isexceptionallyvariable,whichmaybeinfluencedby

differ-entfactors,suchasthehaplotypesofthe␤S-globingene,the

presenceofalpha-thalassemia,andthefetalhemoglobinlevel

(HbF).1,2

Haplotypesofthe␤S-globingenearedeterminedbyspecific

polymorphismpatterns inthe ␤gene complex.They

origi-natefromdifferentregionsofAfrica,fromwhichtheyreceive

theirdenominations:CARorBantu(CentralAfricanRepublic),

Benin(WestAfrica),Senegal(WestAfrica), Cameroon(West

Africa)andSaudi-Indian(Asia,IndiaandtheArabian

Penin-sula). Polymorphism sets not recognizedbythese classical

patternsarereferredtoasatypical(ATP)andoccuratarate

of5–10%.5,6 _{InBrazil,duetotheforcedmigrationofAfrican}

descendants,CARandBenin(BEN)haplotypesarethemost

common.5,7–11

Alpha-thalassemiaisthe resultofdeficientsynthesisof

␣globin chainsinhemoglobin(Hb).The␣ chaingenesare

locatedonchromosome16,andnormalindividualshavetwo

␣genesoneachchromosomewiththisgenotypebeing

rep-resented as ␣␣/␣␣.12 _{The ␣3.7 deletion, which causes the}

mostcommon formofalpha-thalassemiainBrazil,13_isthe

resultofanunequalcross-overexhibiting alossofa3.7kb

DNA fragment.14 _{Recent Brazilian studies show different}

prevalencesforthe␣3.7genotypeaccordingtothe

geograph-ical ancestry of the individuals studied: 4.5% in European

and 21.5% inAfrican descendants,15 _{data similar to those}

obtainedinpopulationsofAfricandescentsbySonatietal.16

Brazilianstudiesanalyzingalpha-thalassemiainindividuals

withHb SS present prevalencesof ␣3.7heterozygous

indi-viduals offrom 17.6 to28.2%, accordingto the geographic

region.9–11,13,17

TheinteractionbetweenHbSSandalpha-thalassemiais

associatedwith an inhibition of Hb S polymerization2,18,19

whichdecreaseshemolyticepisodes2,18_{entailingreductions}

in mean corpuscular volume (MCV) and mean

corpus-cular hemoglobin (MCH) compared to Hb SS individuals

without alpha-thalassemia.20 _{An improvement in the}

patients’ clinical state is observed with reductions in

sickle cell crises.18,21 _{The presence of alpha-thalassemia}

is also significantly associated with a reduction in

white blood cell (WBC)15 _{and reticulocyte}2,17 _{counts, and}

increased levels of Hb A2.17,22–25 Although there is no

defined relationship, increased levels of Hb F are also

reported.2,24

Theobjectiveofthisstudywastoassesstheprevalenceof

␤S-globinhaplotypesandalpha-thalassemiainagroupof

chil-drenwithHbSSandtheirinfluenceonhematologicalprofile

andclinicalmanifestations.

Methods

Thiswasacross-sectionalstudywithconveniencesampling

involving117of122under6-year-oldchildrenwithHbSS,no

mattertheseverityofthedisease,attendedatthePediatric

HematologyOutpatientClinicoftheEscolaPaulistade

Medi-cina,Universidade FederaldeSãoPaulo.Thesampleswere

collectedfromSeptember2007toDecember2009;theage

var-iedfrom3to71months(medianageof35.5months)and62

(52.99%)ofthechildrenweremale.Thestudywasapproval

bytheResearchEthicsCommitteeoftheEscolaPaulistade

Medicina,UniversidadeFederaldeSãoPaulo.

Exclusioncriteria

Patientswhohadreceivedredbloodcelltransfusionsinthe3

monthspriortosamplecollectionandthosereceiving

hydrox-yureawereexcludedfromthestudy.

Clinicalandlaboratorialvariables

Clinical and laboratorialvariables were obtained from the

patient’smedicalrecords.Thefollowinglaboratorialcriteria

wereanalyzed:Hblevel,hematocrit(Ht),redbloodcell(RBC),

WBC,MCV,MCH,reticulocyte,andplateletcounts,andHbF

levels.

Theclinicalvariableswereanalyzedconsideringwhether

the patients had <3 or ≥3 types of manifestations per

year includingvaso-occlusive crisis(VOC),acute chest

syn-drome/pneumonia(ACS/PNM),infections(acuteosteomyelitis

and urinary tract infection) and episodes of acute splenic

sequestration.

Molecularanalysis

DNA was extracted from WBCs in peripheral blood using

phenol–chloroform extraction and ethanol precipitation.26

The Hb S mutation was confirmed by polymerase chain

reaction-restriction fragment length polymorphism

(PCR-RFLP).27 _{Single-tube multiplex-PCR was used for}

alpha-thalassemiagenotypingusingprimersstandardizedbyChong

etal.28_{Determinationofthehaplotypesofthe␤}s_gene

com-plexwasperformed byPCR-RFLPanalysisusingprimersas

proposedbySuttonetal.29_{Alltestswereconductedusinga}

thermalcyclerfromVeritiAppliedBiosystems®_(FosterCity,

CA,USA).

Statisticalanalysis

Non-parametrictestswereusedforstatisticalanalysistaking

intoaccountthevariabilityanddistributionofthestudy

vari-ables.Kruskal–WalliswiththeDunntestwasusedtocompare

morethantwosamples,andtheMann–Whitneytestfortwo

independentsamples.Significancewassetforalphaerrorsof

5%(p-value<0.05);statisticallyanalysiswasperformedusing

theInStat-2(GraphPad®_{,SanDiego,CA,USA)andSigmaStat}

T able 1 – Hema tological da ta accor ding to ␤ S-globin haplotype in 117 sic kle cell anemia childr en. C AR/C AR C AR/BEN BEN/BEN Rar e/A TP p -v alue a n Median (r ang e) n Median (r ang e) n Median (r ang e) n Median (r ang e) Hemo globin (g/dL) 40 8.0 (6.5–10.3) 41 8.0 (7.0–9.9) 13 8.0 (7.2–10.1) 20 8.5 (7.2–9.5) 0.100 Hematocrit (%) 39 22.0 (18.4–30.7) 38 23.7 (20.0–29.4) 11 23.0 (20.5–29.2) 19 24.0 (20.1–29.0) 0.225 RBC ( × 10 12/L) 28 2.6 (1.5–4.0) 30 2.6 (1.8–4.4) 8 2.8 (2.3–3.6) 15 2.7 (2.2–3.5) 0.872 MCV (fL) 36 83.9 (59.6–95.0) 36 80.7 (59.0–89.2) 11 86.6 (75.7–94.2) 18 80.7 (73.0–89.1) 0.015 b MCH (pg) 36 29.2 (20.0–35.2) 35 27.8 (22.1–31.7) 11 28.9 (24.2–33.0) 18 28.2 (24.0–31.0) 0.300 WBC ( × 10 9/L) 40 19.0 (7.0–30.3) 39 16.4 (7.4–32.9) 11 17.8 (5.7–27.7) 20 18.9 (6.9–27.3) 0.820 Platelets ( × 10 9/L) 39 399 (125–795) 39 338 (134–932) 11 440 (189–572) 20 412 (148–770) 0.989 Reticuloc ytes (%) 22 10.9 (0.7–14.7) 28 9.5 (1.5–23.2) 8 8.5 (2.9–20.1) 13 8.8 (1.9–17.8) 0.863 Hemo globin F (%) 30 14.6 (1.8–39.1) 33 17.6 (1.8–51.3) 9 23.4 (10.3–45.6) 17 18.3 (3.7–41.3) 0.403 C AR: Centr al African Re pub lic; BEN: Benin; Rar e: Sene g al, and Saudi; A TP: atypical; RBC: re d b lood cells; MCV : mean corpuscular v olume; MCH: mean corpuscular hemo globin; WBC: white b lood cells. a Kruskal–W allis. b Dunn test: significance observ ed betw een C AR/BEN and BEN/BEN .

Results

Thefrequenciesofhaplotypeswere57.26%forCAR,33.33%

forBEN,8.12%forATP,0.85%forSAUDIand0.43%for

Sene-gal.The genotypedistribution wasCAR/BEN in43 (36.75%)

patients,CAR/CARin41(35.04%),Rare/ATPin20(17.09%)and

BEN/BENin13(11.11%).Inthe20Rare/ATPpatients,the

follow-ingcombinationswereidentified:BEN/ATPineightpatients

(6.84%), CAR/ATP inseven(5.98%), ATP/ATP intwo (1.71%),

andoneindividual(0.85%)withtheSEN/CAR,SAUDI/CAR,and

SAUDI/BENhaplotypes.

Even though the CAR/CAR group presented lower

lev-els ofHb Fthan the other groups, nostatistical difference

was observed. MCV was different between the CAR/BEN

and BEN/BEN haplotypes (p-value=0.01) (Table 1). A

com-parison betweendifferenthaplotypesshowednodifference

concerningthenumberofclinicalmanifestationspresented

(p-value=0.22)(datanotshown).

In relation tothe occurrenceof alpha-thalassemia, two

patients(1.71%)werehomozygousforthe␣3.7deletionand14

(11.97%)heterozygous,totaling13.68%ofalpha-thalassemia

individuals(16/117).Thealpha-thalassemiagroup

(homozy-gotesandheterozygotes)presentedsignificantreductionsin

MCV, MCH and reticulocyte counts, and had a significant

increase in the RBC countcompared tothe group without

thedeletion.AlthoughHbFlevelsinthealpha-thalassemia

group were higher in relationtothe group without

alpha-thalassemia(18.3%;range:4.2–31.1vs.17.8%;range:1.8–51.3,

respectively) no significance was detected (p-value=0.84)

(Table 2). Regarding the number ofclinical manifestations,

there was nosignificant difference betweenthe groups (p

-value=0.66).

Table3showsthefrequencyofalpha-thalassemiain

rela-tiontothe␤S-globinhaplotypes.Significantdifferenceswere

observedinMCVvaluesbetweenindividualswithand

with-outalpha-thalassemiaintheCAR/CARandCAR/BENgroups

(p-value=0.002and0.017,respectively)(Table4).

Discussion

␤S-globin gene haplotypes are being studied in Brazil and

in other countries with the objective of determining their

frequency in different regions and their influence on the

clinical manifestations ofHb SS, since this isnot yet well

characterized.2_{Alpha-thalassemiahasalsobeenconsidered}

an importantmodifying factorofclinical severityinHb SS

asitreduceshemolyticepisodesandaltersthehematological

profileofpatients.18

InpatientswithHbSS,theelevatedconcentrationofHb

Fprotectsagainstvaso-occlusiveevents,consequently

reduc-ingclinicalmanifestations.Steinberg30_{reportedthatHbFis}

themostpowerfuldiseasemodifierandPowars1_statedthat,

inbeingrelatedtolowHbFlevels,theCARhaplotypeis

associ-atedwithmoresevereclinicalmanifestations,whileSENand

SAUDIhaplotypespresenthighHbFlevelswithmilderclinical

manifestations,andtheBENhaplotypepresentsintermediate

Table2–Hematologicaldataexpressedasmedians(range),accordingtopresenceof␣3.7thalassemiain117sicklecell anemiachildren.

␣3.7thalassemia p=(<0,05)a

Absence Presence

n Median(range) n Median(range)

Hemoglobin(g/dL) 98 8.5(6.5–10.5) 16 8.5(7.1–9.5) 0.637

Hematocrit(%) 98 22.0(14.7–30.7) 11 22.6(20.1–28.7) 0.471

RBC(×1012/L) 72 2.6(1.8–4.4) 10 3.0(2.5–4.0) 0.040

MCV(fL) 90 83.5(59.0–95.0) 12 74.4(62.9–83.7) 0.0001

MCH(pg) 89 28.8(22.1–35.2) 12 26.3(20.0–28.7) 0.002

WBC(×109/L) 99 18.1(5.7–32.9) 12 17.2(7.0–21.8) 0.608

Platelets(×109/L) 98 390(125–932) 12 396(285–591) 0.795

Reticulocytes(%) 64 9.8(1.5–22.0) 8 4.1(0.7–13.2) 0.021

HemoglobinF(%) 77 17.8(1.8–51.3) 11 18.3(4.2–31.1) 0.874

␣3.7thalassemia:heterozygousandhomozygous;RBC:redbloodcells;MCV:meancorpuscularvolume;MCH:meancorpuscularhemoglobin;

WBC:whitebloodcells.

a _{Mann–Whitney.}

Table3–Frequencyofalpha-thalassemiaaccordingto ␤S-globinhaplotypesin117childrenwithsicklecell anemia.

Haplotypes ␣␣/␣␣ ␣3.7thalassemia

n(%) n(%)

CAR/BEN 37(31.62) 6(5.13) CAR/CAR 35(29.91) 6(5.13)

BEN/BEN 11(9.4) 2(1.71)

CAR/ATP 6(5.13) 1(0.85)

BEN/ATP 8(6.84) 0

ATP/ATP 2(1.71) 0

SAUDI/CAR 1(0.85) 0

SAUDI/BEN 1(0.85) 0

SEN/CAR 0 1(0.85)

Total 101(86.32) 16(13.68)

␣3.7 thalassemia: homozygousand heterozygous; CAR: Central AfricanRepublic;BEN:Benin;SEN:Senegal;SAUDI:Asia,Indiaand theArabianPeninsula;ATP:atypical.

In the current study, the CARhaplotype was the most

commonhaplotypeencountered,followedbyBEN;thesedata

are similar toother studies conductedinthe southeast of

Brazil.5,7–11_{Despitethefactthattherewasnosignificant}

dif-ferenceinHbFlevelsamonghaplotypes,thesevaluesfollow

thepatterndescribedintheliterature,thatis,lowervaluesin

CAR/CARandCAR/BENgenotypes,followedbyBEN/BEN.7,10

Nosignificantdifferencewasobservedinthenumberof

clinicalmanifestationsbetweenthehaplotypetypes,

proba-bly duetothefact thatthestudiedpopulationpresenteda

mean ageof37months,whenHb Flevelsarestillelevated

inHbSSindividuals,31_{andmanyclinicalmanifestationsare}

not apparentatthis age. Another possiblefactor mightbe

themiscegenationoftheBrazilianpopulationandconsequent

elevatednumberofindividualswithheterozygoushaplotypes.

AstudyconductedbySilva Filhoet al.11_{withasimilarage}

group,encounteredsimilarresults.

TheRare/ATPgroupconsistsofdifferenthaplotype

com-binations, mostly involving CAR haplotype heterozygotes

(11/25=44%)ratherthanheterozygotesandhomozygoteswith

theATPhaplotype.AccordingtoZagoetal.,6_{ATPhaplotypes}

canbeconsideredavariationoftheBantuhaplotype,andthus

wecansupposethattheymightberelatedtogreaterseverity

ofthedisease.Althoughtherarehaplotypes(SENandSAUDI)

are associated with a milder clinical course, they did not

influencetheresultsobtainedastheywereidentifiedinonly

threepatients,confirmingtheirlowprevalenceinthestudy

region.7

Somehematologicalparametersarerelatedtotheseverity

of the disease and are directly implicated in the

phys-iopathology of Hb SS, hence the importance of assessing

their association with haplotypes and alpha-thalassemia.

Inthe comparisonbetweenthe differenthaplotypegroups,

the CAR/CARgroup presentedsignificantlylower Hblevels,

Table4–Meancorpuscularvolume(MCV)presentedasmedian(range)inCAR/CARandCAR/BENhaplotypesaccording tothepresenceof␣3.7thalassemia.

Haplotypes ␣3.7thalassemia p-valuea

Absence Presence

n Median(range) n Median(range)

CAR/CAR 30 85.3(68.5–95.0) 6 77.4(59.6–79.6) 0.002

CAR/BEN 32 81.4(59.0–89.2) 3 71.1(67.2–75.1) 0.017

␣3.7thalassemia:homozygousandheterozygous;CAR:CentralAfricanRepublic;BEN:Benin.

followedbytheCAR/BEN.Thesedataaresimilartoresultsby

SilvaFilhoetal.11_{anddeNeonatoetal.}24

LowMCVand MCHvaluesare associatedwitha

reduc-tioninthesynthesisof␣chainsandrelatedtotheeffectthat

alpha-thalassemiaexertsonthepolymerizationofHbSand

onthedeformityofsickledcells,makingthemlessdense.20

Otherthanthis,thereisevidencethatmicrocytosis,perse,

exert beneficial effects on vaso-occlusive complications in

HbSS.2,21

Thefrequency ofalpha-thalassemia inthe study

popu-lation was similar tothat found inother Brazilianstudies

performedonpatientswithHbSS.9,10,17

Hbvaluesdidnotdifferbetweenthegroupswithor

with-outalpha-thalassemia,similartotheresultsreportedbySilva

Filho et al.11 _{and Stevens et al.}23 _{Significant reductions in}

MCVandMCHlevelsandreticulocytecounts,andasignificant

increaseintheRBCcountin␣3.7deletionpatientsconfirms

dataintheliterature.2,17,22_{Therewasnostatistically}

signifi-cantdifferenceintheWBCcountbetweenthegroups,contrary

tootherpublications thatreportedareductionintheWBC

countinindividualswithalpha-thalassemia.17,22

HbFlevelsdidnotshowanysignificantdifferencebetween

the two groups, similar to results obtained by Silva Filho

etal.,11_{Belisárioetal.}17_{andMouéléetal.,}22 _instudies

con-ductedwithsimilaragegroups.

Thecurrentstudydidnotobservedanyinfluenceof

alpha-thalassemia on clinical manifestations, different to other

studieswherethepresenceofalpha-thalassemiapreserved

splenicfunction,leading topersistenceofsplenomegaly in

patientswithhomozygous␣3.7deletion.31_{ThefactthatHbF}

isstillelevatedinthestudygroupmightalsojustifythelackof

influenceofthedeletioninalpha-thalassemiaontheclinical

manifestationsofthesepatients.

Onefactorthatmightbeconsideredasabiasinthisstudy

isthatdatacollectionofclinicaleventswasfromthepatients’

medicalrecords,whichcouldbeconsidereddeficient.

How-ever the patients were followed-up in the institution, not

only in the outpatient clinic but also in the emergency

wardandduringhospitalization,whichreducesordiscards

biascausedbydependenceonthememoryofthepatient’s

family.

Conclusions

Thefrequencyofalpha-thalassemiainthisstudywassimilar

topreviousstudiesinBrazil,withtheCARhaplotypebeingthe

mostcommon,followedbytheBENhaplotype.The␤S-globin

genehaplotypesandthepresenceofalpha-thalassemiadid

notinfluencethelevelsofHbForthenumberofclinical

man-ifestationspresented.Webelievethataprospectivefollow-up

ofthesepatientswillprovidemoreknowledgeonthe

influ-enceofalpha-thalassemiaandthe␤S-globingenehaplotypes

onclinicalmanifestationsinHbSS.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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