A combination of the α3 7 and MEDII alleles causing hemoglobin H disease in a Brazilian patient B C A h RQ1 G U a A R A A I A d h w t a h a o i t t a f i ( b t j C h 1 T 1 2 3 4 5 6 7 8 9 10 11 12 13[.]
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w w w r b h h o r g
Roberta Dorta Ferreira, Natália de Oliveira Mota, Elza Myiuki Kimura,
Q1
Gisele Audrei Pedroso, Maria de Fatima Sonati∗
a r t i c l e i n f o
Received25November2016
Accepted1December2016
Availableonlinexxx
Introduction
Alpha-thalassemiaisahereditarydiseasewithaworldwide
distributioncharacterizedbyreducedorabsentsynthesisof
hemoglobin␣chains.Deletionsinvolvingthe␣globingenes,
whichareduplicated(␣2 and␣1)andlocatedinthe ␣
clus-ter(16p13.3),arethemostcommoncausesofthediseaseand
accountforover80%ofcases.Lossofafunctional␣geneinthe
haploidgenomeresultsin␣+-thalassemia,whichcanoccurin
aheterozygous(-␣/␣␣)orhomozygous(-␣/-␣)state,whileloss
ofboth␣genesresultsin␣0-thalassemia,whichcanalsooccur
inaheterozygous(–/␣␣)orhomozygous(–/–)state.Afifth
␣-thalassemicgenotypeistheresultofthecombinationofboth
the␣0 and ␣+ alleles (-␣/–).Whilethe firstthreegenotypes
areassociatedwithminimalhematologicalchangesandthe
fourthresultsinhemoglobin(Hb)Bart’shydropsfetaliswith
intrauterineorneonataldeath,thedoubleheterozygous␣0/␣+
(-␣/–)stateleadstoHbHdisease.Thislatterischaracterized
byunstablechaintetramers(4),causingchronic,moderate
toseverehemolyticanemiawithmicrocytosis,hypochromia,
jaundiceandhepatosplenomegaly.1,2
Campinas,SP,Brazil
E-mailaddress:sonati@fcm.unicamp.br(M.deFatimaSonati)
Therearesevendeletionsthatusuallyaffectpopulations aroundtheworld:[-␣3.7,-␣4.2,-(␣)20.5,–MED,–SEA,–FIL,–THAI] Themostcommonmethodusedtoscreenforthesedeletions
is multiplex-gap polymerase chain reaction (Multiplex-gap PCR).3Whenthemolecularbasisofthediseasecannotbe iden-tifiedinthisway,multiplexligation-dependentprobe ampli-fication(MLPA)canbeusedtodetectneworraredeletionsin the␣genes,inthewholeclusterandinthe␣-majorregulatory element(MRE)located40kbdownstreamofthegene.1,2
WedescribethecaseofaBrazilianpatientwithHbH dis-easecausedbythecombinationofthe-␣3.7deletion,themost commoncauseof␣-thalassemiainpopulations,andararer
␣0deletionidentifiedonlybyMPLA
Case report
ThiscasestudywaspartofaprojectapprovedbytheResearch EthicsCommitteeoftheUniversidadeEstadualdeCampinas (Unicamp)underreferencenumber918/2007
A31-year-oldwhiteBrazilianmaleofItaliandescentfrom Araraquara, in the state of São Paulo, with a diagnosis of
http://dx.doi.org/10.1016/j.bjhh.2016.12.001
1516-8484/©2016PublishedbyElsevierEditoraLtda.onbehalfofAssociac¸ ˜aoBrasileiradeHematologia,HemoterapiaeTerapiaCelular ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/)
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Table 1 – Hematological data for the family studied.
RBC
(RV–M:4.5–6.1;F:4.2–5.4)
Hb
(RV–M:14–18;F:12–16)
Ht(%)
(RV–M:41–52;F:36–46)
MCV
(RV:80–99)
MCH
(RV:27–32)
RDW(%)
(RV:10–15)
RC(%)
(RV:0.5–2.5)
HbA2(%)
(RV:1.6–4)
HbF(%)
(RV:<2)
RV:referencevalues;RBC:redbloodcellcount(×109/L);Hb:hemoglobin(g/dL);Ht:hematocrit(%);MCV:meancorpuscularvolume(fL);MCH: meancorpuscularhemoglobin(pg);RDW:redcelldistributionwidth(%);RC:reticulocytecount(%)
hypochromicmicrocyticanemiawasreferredtoour
labora-torytoinvestigatethecauseofhisanemia.Cellcountsand
hematologicalindicesweredeterminedusinganautomated
hematology analyzer (Sysmex XE5000, Sysmex, Japan) and
hemoglobinanalysis wascarried out byelectrophoresison
celluloseacetateinalkalineandneutralpHsandby
cation-exchange high-performance liquid chromatography (HPLC)
(Variant IITM, Bio-Rad Laboratories, Hercules, CA, USA) In
additiontothe hemoglobin(Hb)Aand HbA2 fractions, an
HbHfractionwasdetectedaccountingfor4%ofthetotalHb
Thepatient’sparentswereanalyzed,andalthoughtheydid
nothaveanyclinicalcomplaints,bothhadminorhematologic
changessimilartothosefoundin␣-thalassemia
The first molecular analysis consisted of multiplex-gap
PCR,3whichshowedapatterninthepatient’ssampleobserved
whenthe␣3.7deletionisinahomozygousstate,aresultthat
wouldnotexplaintheHbHdisease.Thesampleswerethen
analyzedbyMLPAusingtheSALSAMLPAP140C1HBAkit(MRC
Holland,Holland),4whichanalyzes approximately360kbof
DNAextendingfrom the16ptelomericregiontotheDECR2
gene.ThefragmentswerecomparedinCoffalyser.Netto
iden-tifypossiblechangesinthenumberofcopiesofthe␣alleles
In addition tothe -␣3.7 deletion, MLPAdetected a large
deletionofapproximately30kbaffectingthe,,␣2,␣1,
␣2 and ␣1 genes (Figure 1A) The extent of this deletion
andthe genesaffected are compatiblewith twopreviously
describeddeletions:–DUTCH,describedinindividualsofDutch
origin,5and–MEDII,describedinindividualsofMediterranean
origin.2,6 These can bedistinguishedfrom each other by a
specificPCRprotocol.5Here,thepatient’sDNAwasfirst
ampli-fied with primers far from the deletion breakpoints, and
the product was re-amplified by nested PCR with primers
flankingthe breakpoints ofbothdeletions With the–MEDII
deletion, fragments of approximately 1.35 and 1.75kb are formed(Figure1C),whilewiththe–DUTCHdeletion,the frag-mentsare1.03and1.41kbinsize.Ourresultsindicatethatthe deletioninquestionconsistsofthe–MEDIIdeletion(Figure1B)
in combination with the -␣3.7 deletion (-␣3.7/–MEDII) in the patientincombinationwiththenormalallele(–MEDII/␣␣)inthe patient’s father.Thepatient’smother washeterozygousfor the-␣3.7deletion(-␣3.7/␣␣).Thehematologicalandmolecular dataforthefamilyareshowninTable1
Discussion
Alpha-thalassemiasarefrequentlycausedbydeletions.The most common of these is the -␣3.7 deletion, which has a prevalenceof20–25%inAfro-Brazilians.7,8HbHdiseaseis spo-radic inBrazilandisgenerallycausedbyacombinationof the-␣3.7deletionand–MEDI,–SEA or-(␣)20.5deletions.9MLPA, however, hasmadeitpossibletodetectrarer orevennovel
␣0 deletionsand eventhose thatonlyaffect theregulatory element.Inthefamilyanalyzedhere,HbHdiseasewasthe resultofacombinationofthe-␣3.7alleleand–MEDIIdeletion,
ageneticalterationnotpreviouslyreportedintheBrazilianor LatinAmericanpopulation.Thisdeletionislargerthanthe–
MEDI(whichisapproximately17kbofDNA)andremovesthe zetageneinadditiontothealphagenes.Ithasbeenfound
inMediterraneancountries,suchasItaly,Turkey,Greeceand Cyprus.6,10
Here,molecularanalysisusingMLPA,confirmedbyfamilial analysis,playedanimportantroleinthediagnosis.MLPAhas madeitpossibletodetectawiderangeofmutations affect-ingtheglobingenesintheBrazilianpopulationthatarenot detectedbyothertechniques
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1.5
1
0.5
0
463 382364 436 292
184 391
178
346
1.75 kb 1.37 kb
POLR3K - 3 - 463nt HBA - HS40 - 178nt HBA - HS40 - 382nt HBZ region - up - 364nt HBZ region - up - 346nt HBM region - up - 184nt
HBA2 - up - 391nt HBA2 - up - 373nt HBA2 - up - 147nt HBA2 - up - 328nt
HBA1&2 - 1 - 220nt HBA1&2 - 1 - 214nt HBA2 - intr-2 - 160nt HBA2 - intr-2 - 244nt HBA1&2 - 3 - 172nt HBA1 - up - 190nt HBA1 - up - 202nt HBA1 - up - 256nt HBA1 - up - 337nt HBA1 - up - 226nt HBA1 - intr-2 - 165nt HBA1 - intr-2 - 250nt
HBQ1 - 3 - 400nt LUC7L - 5 - 277nt
RGS11 - 10 - 472nt AXIN1 - 11 - 418nt DECR2 - 4 - 262nt Ref
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220
220 277 445 472 418 250
214
214
244 160
165 154 310
283
135
190256
202 226
328
337
400
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A
B
C
Figure 1 – (A) Graph showing the result for the proband generated by the Coffalyser.Net software Thex-axisrepresents the probes and they-axisthe ratio of the intensity of the proband sample to the mean intensity of reference samples A ratio of
1 indicates the presence of both alleles, a ratio of 0.5 the loss of one allele and 0 the loss of that region in both alleles (B) Schematic representation of chromosome 16p13.3 The oval represents the telomeric region, the arrows show the locations
of the probes and the boxes the genes The blue line corresponds to the deleted fragment, the dotted lines denote the first and last deleted probe and the regions between the dotted and dashed lines show where the breakpoints may be (adapted from MRC-Holland, 2014) (C) Agarose gel with the nested-PCR amplified product The 1.75 kb and 1.35 kb bands correspond
to the – MEDII deletion 5 Sample 1 is the molecular weight marker (240 bp ladder); samples 2 and 3 are from the proband, and samples 4 and 5 from his father.
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Financial support
Thisstudywascarried outwithfinancialsupportfromthe
Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo
(FAPESP)(Grantno.2014/00984-3;fellowshipno.2015/21184-8),
theConselhoNacionaldeDesenvolvimentoCientíficoe
Tec-nológico(CNPq)andtheCoordenac¸ãodeAperfeic¸oamentode
PessoaldeNívelSuperior(CAPES)oftheBrazilianMinistryof
Education
Conflicts of interest
Theauthorsdeclarenoconflictsofinterest
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