rev bras hematol hemoter. 2017;39(1):80–83
w w w . r b h h . o r g
Revista
Brasileira
de
Hematologia
e
Hemoterapia
Brazilian
Journal
of
Hematology
and
Hemotherapy
Case
report
A
combination
of
the
-
␣
3.7
and
–
MEDII
alleles
causing
hemoglobin
H
disease
in
a
Brazilian
patient
Roberta
Dorta
Ferreira,
Natália
de
Oliveira
Mota,
Elza
Myiuki
Kimura,
Gisele
Audrei
Pedroso,
Maria
de
Fatima
Sonati
∗UniversidadeEstadualdeCampinas(Unicamp),Campinas,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received25November2016 Accepted1December2016 Availableonline28December2016
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␣0and ␣+ alleles (-␣/–).While thefirstthreegenotypes areassociatedwithminimalhematologicalchangesandthe fourthresultsinhemoglobin(Hb)Bart’shydropsfetaliswith intrauterineorneonataldeath,thedoubleheterozygous␣0/␣+
(-␣/–)stateleadstoHbHdisease.Thislatterischaracterized byunstablechaintetramers(4),causingchronic,moderate toseverehemolyticanemiawithmicrocytosis,hypochromia, jaundiceandhepatosplenomegaly.1,2
∗ Correspondingauthorat:DepartmentofClinicalPathology,SchoolofMedicalSciences,UniversidadeEstadualdeCampinas(Unicamp),
Campinas,SP,Brazil.
E-mailaddress:[email protected](M.deFatimaSonati).
Therearesevendeletionsthatusuallyaffectpopulations aroundtheworld:[-␣3.7,-␣4.2,-(␣)20.5,–MED,–SEA,–FIL,–THAI]. Themostcommonmethodusedtoscreenforthesedeletions ismultiplex-gappolymerasechainreaction(PCR).3Whenthe molecularbasisofthediseasecannotbeidentifiedinthisway, multiplexligation-dependentprobeamplification(MLPA)can beusedtodetectneworraredeletionsinthe␣genes,inthe wholecluster andinthe␣-majorregulatoryelement (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
revbrashematolhemoter.2017;39(1):80–83
81
Table1–Hematologicaldataforthefamilystudied.
Familymember Proband Father Mother
RBC
(RV–M:4.5–6.1;F:4.2–5.4)
5.55 6.39 4.74
Hb
(RV–M:14–18;F:12–16)
9.2 13.8 12.5
Ht(%)
(RV–M:41–52;F:36–46)
16.6 44.0 38.6
MCV (RV:80–99)
61.6 81.4 68.9
MCH (RV:27–32)
16.6 21.6 26.4
RDW(%) (RV:10–15)
25.5 14.5 15.3
RC(%) (RV:0.5–2.5)
2.77 1.49 0.98
Hbpattern A2,A,H A2,A A2,A
HbA2(%)
(RV:1.6–4)
1.5 2.70 2.40
HbF(%) (RV:<2)
0.5 0.20 0.20
HbH(%) 4.0 -
-Heinzbodies/HbHbodies Positive Negative Negative
␣genotype -␣3.7/–MEDII –MEDII/␣␣ -␣3.7/␣␣
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 compatiblewithtwo previously 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 mostcommonoftheseisthe-␣3.7deletion,whichhasa preva-lenceof20–25%inAfro-Brazilians.7,8HbHdiseaseissporadic inBrazilandisgenerallycausedbyacombinationofthe-␣3.7
deletionand–MEDI,–SEAor-(␣)20.5deletions.9MLPA,however, hasmadeitpossibletodetectrarerandevennovel␣0 dele-tionsandeventhosethatonlyaffecttheregulatoryelement. Inthefamilyanalyzedhere,HbHdiseasewastheresultofa combinationofthe-␣3.7 alleleand–MEDIIdeletion,agenetic alteration not previouslyreported inthe Brazilian or Latin American population.Thisdeletionislargerthan the–MEDI (whichisapproximately17kbofDNA)andremovesthezeta gene in addition tothe alpha genes. It hasbeen foundin Mediterranean countries, such asItaly, Turkey, Greece and Cyprus.6,10
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revbrashematolhemoter.2017;39(1):80–831.5
1
0.5
Ratio
0
463 382364 436 292
184 391
178
POLR3K HS - 40
HBZ HBZP HBZ2P HBA1P HBQ1 LUC7L ITFG3 RGD11
HBA2
HBA1
346
1 2 3 4 5
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
HBA1 - 3 - 154nt
HBA1 - do
wn - 283nt
HBZP1 region - do
wn - 292nt
HBZ - 1 - 436nt
HBA1 - do
wn - 310nt
HBQ1 - 3 - 400nt LUC7L - 5 - 277nt
ITFG3 - intr 01 - 445nt
RGS11 - 10 - 472nt AXIN1 - 11 - 418nt DECR2 - 4 - 262nt Ref
erence* - 238nt
Ref
erence* - 481nt
Ref
erence* - 409nt
Ref
erence* - 269nt
Ref
erence* - 130nt
Ref
erence* - 141nt
Ref
erence* - 196nt
Ref
erence* - 355nt
Ref
erence* - 208nt
Ref
erence* - 300nt
Ref
erence* - 454nt
HBA2 CS – 3 (MUT) CS) - 136nt
147
373 220
220 277445472418
250 214
214
244 160
165 154 310 283
172 172
135 190256
202 226
328
337
400
262
AXIN1 DECR2
A
B
C
Figure1–(A)GraphshowingtheresultfortheprobandgeneratedbytheCoffalyser.Netsoftware.Thex-axisrepresentsthe
probesandthey-axistheratiooftheintensityoftheprobandsampletothemeanintensityofreferencesamples.Aratioof
1indicatesthepresenceofbothalleles,aratioof0.5thelossofonealleleand0thelossofthatregioninbothalleles.(B)
Schematicrepresentationofchromosome16p13.3.Theovalrepresentsthetelomericregion,thearrowsshowthelocations
oftheprobesandtheboxesthegenes.Thebluelinecorrespondstothedeletedfragment,thedottedlinesdenotethefirst
andlastdeletedprobeandtheregionsbetweenthedottedanddashedlinesshowwherethebreakpointsmaybe(adapted
fromMRC-Holland,2014).(C)Agarosegelwiththenested-PCRamplifiedproduct.The1.75kband1.35kbbandscorrespond
tothe–MEDIIdeletion.5Sample1isthemolecularweightmarker(240bpladder);samples2and3arefromtheproband,and
revbrashematolhemoter.2017;39(1):80–83
83
Financial
support
Thisstudy wascarriedout withfinancialsupportfromthe 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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