Rev. Bras. Hematol. Hemoter. vol.38 número1

rev bras hematol hemoter. 2016;38(1):79–81

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

Revista

Brasileira

de

Hematologia

e

Hemoterapia

Brazilian

Journal

of

Hematology

and

Hemotherapy

Case

Report

RHD*weak

D

type

38

:

a

family

study

Sidneia

Sanches

Costa

,

Akemi

Chiba

,

Bruno

Cruz

,

Dante

Langhi

Júnior

,

José

O.

Bordin

a_{UniversidadeFederalSãoPaulo(UNIFESP),SãoPaulo,SP,Brazil}

b_{FaculdadedeCiênciasMédicasdaSantaCasadeSãoPaulo(FCMSCSP),SãoPaulo,SP,Brazil}

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Articlehistory:

Received2September2015 Accepted18October2015

Availableonline21November2015

Introduction

TheRHDgeneisresponsiblefortheexpressionoftheDantigen whileRHCEencodesproteinscarryingantigensCorc,andE ore.RHDispresentorabsentdependingontheRHhaplotype, andRHCEdisplaysfourcommonalleles(ce,Ce,cE,CE) respon-siblefortheexpressionofthetwoantithetical(allelic)series ofantigens.RHDgenedeletionistheleadingcauseofthe D-phenotypeworldwide.1_{Functional(antigen)variationsinthe}

Rhblood groupsystemisdeterminedbyinsertion/deletion, singlenucleotidepolymorphisms(SNPs),andgeneconversion eventsintheRHDandRHCEgenes.AweakDtypeisavariant oftheRhDproteinthatcomprisesanaminoacidsubstitution locatedinthetransmembraneousorintracellularsegments andexpressesareducedamountoftheDantigen[generally lessthan5000Dantigensperredbloodcell(RBC)].2,3

TheRHD*weakDtype38isproducedbythesinglenucleotide

change(833G>A)intheexon6,causingaGly278Asp substitu-tioninthetransmembraneousRhDprotein.ThisRHDallele showsreducedexpressionoftheDantigenontheRBC sur-faceandmaybeerroneouslytypedasDnegativebystandard serologicmethods.4–6_{ThefrequenciesofweakDtype38vary}

indifferentpopulations.TheweakDtype38was foundin

∗ _{Correspondingauthorat:RuaDiogodeFaria,824,04023-061SãoPaulo,SP,Brazil.}

E-mailaddress:[email protected](J.O.Bordin).

1.5%ofCaucasianswiththeCceephenotype,4_whilea

rela-tivelyhigherfrequencyof2.6%wasrecentlydetectedamong BrazilianswiththeCceephenotype.6_{AlthoughtheRHD*weak}

Dtype38isconsideredaveryrareallele,itisrelatively com-moninthePortuguesepopulation.7_{Inthisstudyweanalyzed}

threegenerationsofafamilywithRHD*weakDtype38using serologicandmolecularmethods.

Case

report

Familystudyandbloodsamples

Asamplefroma45-year-oldfemaleblooddonorwastestedD negativebyroutineserologictests(Cceephenotype)but posi-tivebytheadsorption-elutiontechnique.Inordertoelucidate theseresultsweexaminedbloodsamplesfrom12members ofherfamilywereexamined.

Immunohematologic serological tests were performed using commercial monoclonal antibodies (MoAbs) MS-26+TH-28 and ESD1 by classical serology techniques, hemagglutinationingelcards(DiaMed,LatinoAméricaS.A.) andbythetubemethodswithanti-DMoAbsIgM(HM10,P3X61, P3X21211F1,P3X21223B10;Diagast,Loos,France)andanti-D

http://dx.doi.org/10.1016/j.bjhh.2015.10.001

80

II-1

III-1

dce/dce Weak D type_{38 Ccee} III-2 dce/dce

II-2 II-3

III-3

DCcee DCcee

III-4

II-4 II-5

DCcee Weak D type 38

Ccee

Weak D type 38 Ccee Weak D type 38

Ccee Weak D type 38

Ccee Weak D type 38

Ccee Weak D type 38

Ccee

I-2

Figure1– PedigreeoffamilywithRHD*weakDtype38.

MoAbsIgG usingtheindirect antiglobulintest(IAT) (HM16, P3X35, P3X241, P3X249, P3X290; Diagast, Loos, France). Rh phenotypeswereperformedbyhemagglutinationingelcards, accordingtothemanufacturer’sinstructions(DiaMed,Latino AméricaS.A.)usingpolyclonalantibodiesagainstD,C,c,Eand e.

TheDantigendensityofsampleswasdeterminedbyflow cytometry(FACSCalibur, Becton Dickinson,Heidelberg, Ger-many) according to a previously described protocol8 _with

sevenanti-DIgGMoAbs,HM16,P3X35,P3X241,P3X249,P3X290 (Diagast,Loos, France)and MS26and ESD1(DiaMedLatino AméricaS.A.).

Bloodsamplesfromfamilymemberswereinvestigatedby polymerasechainreaction(PCR)withspecificprimersfortwo genomicregionsoftheRHDgene,intron4andexon10andall tenRHDexonsweresequencedfulllength.9,10

WefoundsevenmembersofthisfamilytestedDnegative byroutineserologictests(Ccee phenotype)and positiveby theadsorption–elutiontechnique.TheirRBCsreacted nega-tivelytofourIgManti-D,butshowedweaklypositiveresults withfourIgGanti-Dreagents(P3X249,P3X35,P3X241,HM16) (Table1).Theflowcytometricanalysisofsamplesfromthese sevensubjectsshowedDantigendensitiesrangingfrom60 to80sitespercell.Molecularanalysisidentifiedthepresence

ofRHD*weakDtype38inthreegenerationsofthisBrazilian

family(Figure1).

Discussion

Thisstudyrevealedmaternalinheritance(firstgeneration)of

theRHD*weakDtype38withtheCceephenotypebyherfive

Table1–Patternofreactivityseenwithdifferentclones ofanti-DantibodiesandDantigendensitiestested againstredbloodcellsfromindividualswithRHD*weak Dtype38.

Anti-DMoAb Depitope Isotype Reactivity

P3X249 2.1 G (±)

P3X290 3.1 G (−)

ESD1 4.1 G (−)

P3X35 5.4 G W

P3X241 5.4 G (±)

HM16 6.4 G W

P3X61 6.4 M (−)

HM10 6.6 M (−)

P3X21211F1 8.2 M (−)

P3X21223B10 9.1 M (−)

MS26 9.1 G (−)

DAntigendensitiesa _{60–80sites/cell}

MoAb:monoclonalantibody;(−):negativeresult;W:weakresult; (±):veryweakresult.

a _{Dantigendensitiesdeterminedbyflowcytometrywithanti-DIgG}

monoclonalantibodies.

childrenandthesecondgenerationshowedpaternal inheri-tancebyason.ThisfamilyevaluationshowedthatRHD*weak Dtype38wasinheritedasanancestralallele.

Inconclusion,thisstudyfoundarelativelyhighprevalence

ofRHD*weakDtype38inBrazilians,rememberingthatBrazil

was colonized by the Portuguese.6,7 _{This isthe first report}

revbrashematolhemoter.2016;38(1):79–81

81

DnegativebystandardserologicmethodsincludingIAT.Thus, thiscouldrepresentapotentialriskinbloodtransfusionsince unitsofbloodcollectedfromtheseindividualscouldbe trans-fusedtoD-negativerecipientscausingRhDalloimmunization.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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