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Hematology OctoberDecember 2006 11(56) 389-398

Duffy blood group and malaria

DANTE M LANGHI Jr amp JOSE ORLANDO BORDIN2

informahealthcare

1Department of Hematology and Transfusion Medicine~ Santa Casa Medical School~sao Paulo~S~ Brazil~ and 2Departmentof Hematology and Transfusion Medicine~ Federal University of sao Paulo - Escola Paulista deMedicina~ sao Paulo~s~ Brazil

(Received 4 October 2005j in final form 18 October 2005)

AbstractVery important progress has been made over the last years in understanding the Duffy blood group system and its complexityThe Duffy blood group antigen serves not only as blood group antigen but also as a receptor for a family of proinflammatorycytokines termed chemokines and as a receptor for Plasmodium vivax malaria parasites The Duffy antigen has been termedthe Duffy Antigen Receptor for Chemokines (DARC) or the Duffy chemokine receptor DARC might playa role as ascanvenger on the red blood cell surface to eliminate excess of toxic chemokines produced in some pathologic situations [48]

Plasmodium vivax (P vivax) causes approximately between 70 and 80 million cases of malaria per year and is the mostamply distributed human malaria in the world [51] Individuals with the Duffy-negative phenotype are resistant to P vivaxinvasion and the molecular mechanism that gives rise to the phenotype Fy(a - b - ) in black individuals has been associatedwith a point mutation - 33TC expressed in homozigosity in the FYB allele [5] Despite P vivax be widespread throughout thetropical and subtropical world it is absent from West Africa where more than 95 of the population is Duffy negativeRecently this point mutation has been described in heterozigosity in the FYA allele in others malaria endemic regions [78]and until now we do not know if it confers a certain degree of protection against P vivax infection

Keywords Duffy~ malaria~ resistance~dose effect

Background

The Duffy blood group system is important in clinicalmedicine because of transfusion incompatibilities andhemolytic disease of the newborn (HDN) [1] It is thenumber 008 in the International Society of BloodTransfusion (ISBT) nomenclature

The product of Duffy (FY) gene is a glycoprotein(gp-Fy) which spans the plasma membrane seventimes and has an extra cellular N-terminal domain andan intracellular C-terminal domain The systemconsists of four alleles five phenotypes and fiveantigens The Duffy locus is located at chromosomel-lq22-23 [2]

Initially two major co-dominant antigens weredescribed and designated Fya and Fyb and codifiedby two co-dominant alleles designated FYA andFYB [3]

Among Caucasians the anti-Fya and anti-Fybantibodies define the phenotypes Fy(a + b - )Fy(a + b + ) and Fy(a - b + ) that most of the time

represent the genotypes FYA FYA FYA FYB andFYBFYB respectively The phenotype Fy(a - b - )is more common among black individuals (AfroAmericans or Occidental Africans) [4] The phenotypeFy(a - b - ) represents the genotype FYB SEFYB SE(SE-silent erythroid) being FYB SE a silent allele atlocus FYAFYB (Duffy) The FYB SE gene in themajority of black individuals that present thephenotype Fy(a - b - ) is an allele that is the samein its structural region as FYB but has a pointmutation in the GATA box promoter region werethe erythroid GATA-l transcription factor adhereschanging the transcription activity of this factorabolishing the gene transcription in erythrocytes ofindividuals that present homozygosity for thismutation It is common among black individuals butrare among other races [5]

In 1965 the FYX or FYB wx (WK- Weak) allele atDuffy locus was described The gene itself does notencode the production of a distinct antigen fromothers of the Duffy system Individuals that inherit

Correspondence J O Bordin Universidade Federal de Sao Paulo Disciplina de Hematologia e Hemoterapia Rua Botucatu 740 Sao PauloSP 04023-902 Brazil Tel 5511 5579 1550 Fax 5511 5571 8806 E-mail jobordinhematoepmbr

ISSN 1024-5332 printlISSN 1607-8454 online copy 2006 Informa UK LtdDOl 10108010245330500469841

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390 D M Langhi amp J O Bordin

the FYB WK gene have erythrocytes that react weaklywith some but not with other examples of anti-Fybantibodies However the Fybwk antigen acts as a Fybantigen of low expression and there is no anti-Fybwk[6] Two mutations in the coding region of the FYBgene are associated with low expression of this alleleMost of the time Fybwk has been described amongCaucasian individuals where its occurrence is notrare

Recently Zimmerman et al [7] described thepresence of the FYA NULL gene in a population of amalarial endemic area at Papua-New Guinea and wehave described the FYA NUL in a population of theAmazon region in Brazil [8]

Another function of the Duffy antigen is being areceptor for the Plasmodium vivax parasite of humanmalaria [9]

E vivax is practically absent in Occidental Africawhere more than 95 of the population is Duffynegative The E vivax and the Plasmodium knowlesineed the interaction with the Duffy antigen tointernalize within the erythrocyte [1011]

The Duffy antigen also acts as a receptor for a familyof pro-inflammatory cytokines called chemokines andis also called Duffy Antigen Receptor for Chemokines(DARC) [12]

Introduction

The Fya antigen was identified serologically in 1950 byCutbush et al [3] during an investigation of atransfusion reaction in the serum of a multiplytransfused patient with hemophilia Anti-Fyb wasdiscovered the following year by Ikin et al [13] Initiallythe antigen with which the serum from the first patientreacted was named Fya and later the determinant geneof this antigen was called FYA On this occasion theexistence of the allele FYB was postulated and was laterdemonstrated by Ikin et al [13]

In Caucasians these antibodies define the pheno-types Fy(a + b -) Fy(a + b +) and Fy(a - b + )that most of the time represent the genotypesFYAFYA FYAFYB and FYBFYB respectively In1955 Sanger et al [4] reported that the phenotypeFy(a - b -) is more common in American Blackindividuals than is any phenotype in which Fya or Fyb ispresent It was thought that the Fy(a - b - )phenotype probably represented the FYB SEFYBSEgenotype with FYB SE being a silent allele at theFYAFYB (Duffy) locus

In blood transfusion Fya and Fyb are the mostimportant antigens in the Duffy system and are fullydeveloped at birth [14]

Anti-Fya is not a particularly uncommon antibodyand is found both as a single entity in the sera ofpersons phenotypically Fy(a - b + ) and as one of amixture of antibodies made by good respondersAnti-Fya is usually an IgG antibody mostly IgG 1

most often immune reacts optimally or only by IATand naturally occurring anti-Fya is rare [15] Anti-Fyahas been incriminated in immediate and delayedhemolytic transfusion reactions [1617] and manycases of hemolytic disease of the newborn (HDN)due to anti-Fya varying from mild to fatal have beenobserved [18]

Anti-Fyb is found twenty times less frequently thananti-Fya [19] and most examples appear to occur inindividuals who make multiple antibodies afterexposure to red cells Contreras et al [20] havedescribed the production of anti-Fyb by a womanexposed to the antigen when the fetus she was carryingwas given an intrauterine transfusion and Issit [21]has described a potent anti-Fyb in the serum of a non-transfused male donor with no known exposure toforeign red cells Thus the antibody appeared to benaturally-occurring

The Fy3 antigen was described in 1971 [22] andFy4 and Fy5 in 1973 [23] In 1987 the Fy6 antigenwas recognized by a murine monoclonal antibody andadded to the Duffy system [10]

In 1965 Chown et al [6] described a new allelenamed FYX or FYB WK as its product was totallyuncertain at the Duffy locus The Fybwk antigenbehaves as a weak Fyb and there is no anti_Fybwk It isinherited as an allele of FYA and FYB co-dominantwith FYA and recessive to FYB This new allele wasconsidered the fourth Duffy allele [6] The gene doesnot encode production of an antigen that is distinctfrom others in the Duffy system Instead individualswho have inherited an FYB WK but not a FYB genehave red cells that react poorly with some and not atall with other examples of anti-Fyb and sometimes theantigen can be detected only by adsorption andelution of anti-Fyb [6] The presence of Fybwk isassociated with diminished expression ofFy3 Fy5 andFy6 antigens markedly in individuals homozygous forFYB WK bind weakly to anti-Fy3 and anti-Fy6 [24]The low expression of Duffy antigens in these cases isdue to a low copy number of the Duffy protein in thecell surface with approximately one-tenth as muchexpression and not due to the conformationalchanges in the protein [25]

The Duffy system is number 008 in the ISBTnomenclature

Duffy protein

The glycoprotein (gp-Fy) that expresses the Duffyantigens was initially described in 1982 by Moore et al[26] and in 1984 Hadley et al [27] demonstrated byimmunodetection technique that the red blood cell(REC) component that carries the Duffy antigens is aprotein with a molecular weight of 35-43 kDasensitive to chymotrypsin and resistant to trypsintreatments The hydropathy map predicts an extra-cellular N-terminal domain of 60 residues seven

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transmembrane a-helices short protruding hydro-philic loops and an intracellular C-terminal domainof 28 residues [28]

The gp-Fy is composed of the Fya and Fyb epitopesin addition to the Fy3 Fy4 Fy5 and Fy6 epitopesThe Fya e Fyb epitopes are located in the extracellulardomain in resides 42 and the Fy3 epitope is locatedon the third extracellular loop The Fy6 epitope hasbeen accurately mapped to a heptapeptide comprisingresidues 19-25 and is located between the twoglycosylation sites [29]

The Duffy protein is encoded by a short single copygene (15 kb 2 exons) located on chromosome 1 andexhibits significant protein sequence homology withthe human and rabbit IL-8 receptors DARC is mostprobably organized in seven transmembrane domainssimilar to other members of the G-protein coupledchemokine receptors [30] The major (336 aa) andminor (338 aa) DARC polypeptide isoforms areencoded by the spliced and unspliced Fy mRNAsrespectively which differ by the sequence of the sixand eight NHTterminal amino acids respectively butbind anti-Fy antibodies and chemokines equally wellDARC has a specialized tissue distribution as presenton RBCs endothelial cells of post-capillary venules(a site of leukocyte trafficking) Purkinje cells of thecerebellum and presumably on some epithelial cells inthe kidney [3132]

Duffy polymorphism

The molecular basis of the Duffy blood grouppolymorphisms has been determined The Duffy(FY) gene is located in the 1q22-q23 region ofchromosome 1 [33] The Duffy cDNA was originallycloned from a non-spliced mRNA and it was assumedthat Duffy is an intron less gene [34] The Duffytranscription unit encompasses 1572 nt includingexon 1 of 55 nt a single intron of 479 nt and exon 2 of1038 nt [35]

Duffy blood group and malaria 391

The Fyab antigenic polymorphism results from apoint mutation at nucleotide 125 in the gp-FY genewith the change of a single base G 125A This changeresults in a single amino acid difference changingglycine by aspartic acid at position 42 of gp-FyGlycine at position 42 (Gly42) defines the Fya antigenand aspartic acid at position 42 (Asp42) defines theFyb antigen [34] (Figure 1)

The molecular mechanism that gives rise to thephenotype Fy(a - b - ) in black individuals has beenclassically associated with a point mutation - 33TC inthe promoter region of a FY B allele that disrupts abinding site for the transcription factor GATA-1 amutation that specifically abolishes the transcriptionalactivity in erythroid cells GATA box of individuals thatexpress this mutation in homozygosity [5] (Figure 2)This mutation defines the FYB SE allele that isstructurally identical to the FYB allele in its codifyingregion

Individuals showing heterozygosity for the GATAbox mutation demonstrate dosage effect and haveapproximately one-half the level of Duffy antigen inerythrocytes [25]

In Caucasians the Fy(a - b -) phenotype isextremely rare and in all cases studied so far it isassociated with mutations or deletions within thecoding sequence of the DARe gene [32] It is assumedthat these mutations in contrast to the erythroidspecific mutation - 33TC should lead to the trueF~ull phenotype with a lack of expression ofDARC inboth erythroid and nonerythroid cells

For individuals from malarial endemic regions insoutheastern Asia the Fy(a - b -) phenotype ispractically nonexistent and the molecular basis of thisphenotype is not the same as that described in Blackindividuals in other words homozygosity for theFYB ES allele [37] In some locations individualshaving the Fy(a - b - ) phenotype possess the FYA-FYA and FYAFYB genotypes without the presence ofthe FYBESFYBES genotype [37] therefore there isheterogeneity in the findings In this group of

Duffy 5Gene

mRNA

Exon 1

------

Promoter region -

5

Exon 2 1572 (nts)

3

gp-Fy NHZ COOHbull42 (residue number)

Fya Gly

Fyb Asp

Figure 1 Schematic representations of the Duffy gene FY (top) mRNA (middle) and gp-Fy (bottom) with polymorphism FYAFYB at nt125 and residues Gly for FYA and Asp for FYB at position 42 (Modified from Pogo Chaudhuri [32])

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392 D M Langhi amp J O Bordin

Point mutationnt-33T -7 C

FYA or FYB Gene

FYBSE Gene

I J

GATA-l

Coding Region 1008

3

Figure 2 Schematic representation of point mutation in GATA-I site responsible for the non-expression of gp-Fy in RBCs of blackindividuals with Fy(a - b - ) phenotype of the mutation point at the bonding site of GAT A-I not the expression of the GPD in RBCs of themajority of Black individuals with the Fy(a - b - ) phenotype (Modified from Issit Anstee [36])

individuals a study was carried out on the phenotypingand genotyping and the presence of the antigen Fyawkwith low reactivity to anti-Fya was observed

The presence of the Fya antigen which is weaklyreactive was also described in Malaysians [38]

Recently Zimmerman et al [7] described thepresence of this mutation linked to the FYA allele in apopulation living in a malaria endemic region of PapuaNew Guinea and this FYA null appears to have a morerecent origin than that of FYB null

We demonstrated the presence of the FYA null allelein blood donors and Individuals from a malarialendemic region of Brazil [8]

The Fybwkphenotype is associated with a missensemutation in the coding region of FYB gene with asingle substitution at nt 265C- T that produces anamino acid change Arg89Cys in gp-Fy Near thismutation another mutation G298A resulting inAlalOOThr amino acid substitution was also ident-ified This mutation is silent and is also present in FYBor FYA alleles from Fy(a - b +) and Fy(a + b + )individuals [25] These amino acid substitutions occurin the first intracellular loop of gp-Fy and only theArg89Cys substitution produces the Fy(a - b + wk)phenotype (Figure 3)

The amino acid substitutions occur in the firstintracellular loop of gp-Fy representing considerable

modification in the chemical nature of the site andresults in very low membrane expression of DARC inFy(a - bweak)erythrocytes entailing a weakening ofthe Fyb antigen [34]

The expression of Fy3 Fy6 and Fyb antigens whenthe G298A mutation is present is similar to thoseencoded by cDNA of FYB contrary to the situationwhen the mutation C265T exists [25]

In non-Ashkenazi Jews individuals with thephenotype Fy(a - b -) show the promoter wild-type GATA however Non-Ashkenazi Jewish indivi-duals with the Fy(a - b -) phenotype weredescribed as presenting the region which promotesthe FYB gene in wild type form but with the C265Tmutation in the codifying region of the FY gene whichalters the antigenic determinants of the DARCweakening the Fyb antigen [39]

Also described were non-Ashkenazi Jewish indivi-duals with the Fy(a - b -) phenotype and theFYBFYB genotype heterozygotes for the GATAmutation entailing simultaneously the C265Tmutation weakener of FYB [39]

Similar to that which was described in non-Ashkenazi Jews was also described in BrazilianBlack individuals namely the association of themutation responsible for the FYB fraco genotype(C265T) with the T-33C mutation (FYB es) in the

Duffy 5Gene

mRNA

Exon 1

------ Promoter 1

region bullbull I

5

Exon 2

1008 (nts)

bullbullbull-3AAAn

gp-Fy NH2 bullbull bullbull89 100 (residue number)

ArgCys Alaffhr

Figure 3 Schematic representation of FY gene with the C265T mutation (FYB WK) encoding the amino acid change Arg89Cys and thesilent mutation G298A codifying the amino acid change AlalOOThr (Modified from Pogo Chaudhuri [32])

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individuals whose Fy(b -) phenotype cannot beexplained by the isolated mutation in the GATA-1box [40]

Functional aspects

Although all blood group antigens are serologicallydetectable on RBCs most of them are also expressedin non-erythroid tissues raising further questions ontheir physiological function under normal andpathological conditions In addition to their structuraldiversity blood group antigens also possess widefunctional diversity and can be schematically sub-divided into five classes (a) transporters and channels(b) receptors for ligands viruses bacteria andparasites (c) adhesion molecules (d) enzymes and(e) structural proteins

Many RBCs surface molecules among those whichcarry blood groups are receptors for viruses bacteriaand parasites suggesting that these antigens may playadirect role in the pathogenesis of infectious diseases[4142]

A variety of microorganisms recognize carbohydratestructures present on glycolipids and glycoproteinsfor instance sialic acids which are abundantlyrepresented on glycophorin A (GPA) or the Cala1-4 Gal motif shared by P Pk and PI glycolipids usedby several bacterial strains and toxins responsible forupper urinary tract infections [43]

The Duffy antigens (Fyab Fy3 and Fy6) are carriedby a membrane glycoprotein exhibiting two interestingbiological properties

bull as a promiscuous receptor for chemokines of theCC (RANTES MCP-1) and CXC (IL8 mgSA)subfamilies of proinflammatory peptides namedaccording to the structure of a conserved cysteine(C) motif Hence the Duffy protein was renamedDARC for Duffy AntigenReceptor for Chemo-kines

bull as erythroid receptor for R vivax and R knowlesi

Duffy antigen receptor for chemokine

The fact that RBCs possess a chemokine receptor wasfirst established by Darbone et al [44]

Chemokines constitute a family of proinflammatorycytokines capable of activating leukocytes and causingchemotaxis but other important functions have beendiscovered including angiogenic and angiostaticactivities [45] The number and spacing of amino-terminal cysteines have been used in the classificationof chemokines into four families C CC CXC andCXXXC The biological effect of chemokines ismediated by the binding and activation of G-protein-coupled seven-transmembrane domain chemokinereceptors [46] Some chemokines (classes CC and

Duffy blood group and malaria 393

CXC) also bind to the Duffy blood group glyco-protein which is now called DARC

Of the cellular chemokine receptors CXCR4CCR5 and Duffy are the only receptors that havebeen unequivocally proven to act as coreceptors forcell entry of pathogens Duffy for the entry of malarialparasite R vivax and CCR5 for the entry of M-tropicstrains of human immunodeficiency virus (HIV) [47]

During experiments with chemokine receptors inerythrocytes the perfect correlation between thebonding of the chemokines to the erythrocytes andthe presence of the Duffy antigen was well established[12] It has been demonstrated that anti-Fy antibodiesinhibited the chemokine binding ability to Duffypositive erythrocytes and chemokines that bind toerythrocytes block RBC invasion by malarial parasitesthat use the Duffy antigen as a receptor [12]

CC and CXC chemokines bind to DARC with highaffinity suggesting some role in inflammatory reactions[31] DARC might play a role as a scavenger on theRBC surface to eliminate excess of toxic chemokinesproduced in some pathologic situations [44]

There are some experimental studies suggestingthat DARC is a redundant protein that may playa rolein the regulation of induced leukocyte trafficking invivo [48]

The CXCR4 and CCR5 chemokine receptors aremandatory cofactors for HIV infection by interactingwith the viral envelope gp 120 in the presence of CD4[49] A report shows that HIV-1 viral particles mightbind to the Fy protein suggesting that RBCs mayfunction as a virus reservoir or as a receptor for theentry in some cells [50]

Duffy antigen receptor for R vivax

R vivax causes approximately between 70 and 80million cases of malaria per year and is the most amplydistributed human malaria in the world [51]

One of the most interesting aspects of the Duffyantigen is its function as a receptor for the humanmalaria parasite R vivax

In 1975 Miller et al [9] showed that Duffy-negative human RBCs were resistant to invasion byR knowlesi a monkey malaria parasite that was knownto be capable of invading human RBCs and in rareinstances of infecting humans Furthermore anti-Fyaand anti-Fyb blocked invasion of R knowlesi intoFy(a + b -) and Fy(a - b +) RBCs respectivelyand the treatment of the RBCs with enzymes whichremove from their surface the antigenic determinantsFya e Fyb (chemotrypsin and pronase) making theRBCs resistant to invasion [9]

Studies on R knowlesi were extended to R vivax ahuman malaria that is second only to Plasmodiumfaleiparum in terms of the toll it takes on populationsof endemic areas Although R vivax is widespreadthroughout the tropical and subtropical world and

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394 D M Langhi amp J O Bordin

is a major drain on the health care resources of Indiaand Southeast Asia it is absent from West Africawhere more than 95 of the population is Duffynegative Miller et al [52] showed that resistance toR vivax correlates with the Duffy-negative pheno-type and more recently Barnwell et al [53] showedthat R vivax merozoites are incapable of invadingDuffy-negative RBCs Thus R vivax like R knowlesirelies on a Duffy antigen-parasite ligand interactionsfor invasion It should be noted that R vivaxpreferentially invades reticulocytes [54] althoughmature RBCs as well as reticulocytes express acoreceptor that along with the Duffy antigen isnecessary for optimal invasion by R vivax Thebonding agent of the R vivax parasite whichspecifically bonds itself to the reticulocytes was alsoidentified and the cDNA encoding this protein wascloned [54]

When the anti-Fy6 antibody became availablestudies demonstrated that the presence of Fy6 is whatresults from the invasion of human RBCs by themerozoites of the R vivax [53]

In humans the presence or absence of Fy6correlates with the presence or absence of the Fyaand Fyb In other words the Fy(a +) or Fy(b + )RBCs are always Fy6 positive and Fy(a - b - ) RBCsare Fy6 negative The invasion of the Fy(a + ) RBCsby R vivax can be partially blocked by the covering ofthe RBC with anti-Fya [9] and totally blocked bycovering the RBC with anti-Fy6 [55]

The fact that the great majority of black African andAfro-American individuals are resistant to infection byR vivax is directly related to the Fy(a - b - )phenotype or in other words the absence of the Fyaand Fyb antigens

One Duffy binding protein of R vivax calledPvDAP-l (Pv - indicates R vivax and DAPindicates Duffy-associating protein) has a criticalrole as a bonder in the adhesion of the parasite andposterior invasion of the positive Duffy RBCs [56] Rvivax as with other species of human Plasmodiuminitiate erythrocyte invasion through the expression ofvarious surface organelles and structures on themerozoite which bind with the surface proteins ofthe erythrocyte The well-characterized interactionof the bonder-receptor involves the Duffy bondingprotein expressed in the merozoite form of theR vivax and its corresponding receptor in theerythrocyte the receptor DARC This interaction isunique among human malaria infections where theinteraction of the bonding receptor is essential to theinvasion of the erythrocyte by R vivax Alternatemodels of erythrocytic invasion have not yet beendescribed [57] This Duffy bonding protein ofR vivax belongs to the family of bonding proteinsfor erythrocytes which also includes the bondingprotein for sialic acid R jalciparum and to the Duffybonding protein R knowlesi

R vivax merozoites invade reticulocytes that expressthe Duffy protein and the counter receptor is a140 kDa protein (PvDBP) that belongs to Plasmodiumadhesion proteins including EBA-175 and PfEMPl(see above) all having in the NHz-ter region acysteine-rich domain (called DPP region II) mediat-ing erythrocyte binding [5859]

It is not known whether the Duffy antigen is astructural junction component because the molecularnature of junction has yet to be determined It is clearhowever that the antigen interaction of the parasiteDuffy-receptor is crucial to the formation of thejunction and subsequent invasion [57]

Several blood group molecules of red cell surfacecontribute to the complex mechanisms of invasion andsequestration steps that characterize the most severeform of malaria caused by R jalciparum R jalciparummerozoites may invade RBCs through sialic acid-dependent and independent pathways [6061] GPA isinvolved in the sialic-acid dependent pathway and thecounter receptor of GPA is the parasite proteinerythrocyte binding antigen (EBA) of 175 kDa [62]which binds to sialic acid and peptide backbone ofGPAthrough a cysteine-rich domain (region II) analogousto the one mediating R vivaxDuffy protein interaction[63] Recently a homologue to EBA-175 calledBAEBL has been shown to use GPC for invasion[64] Rosette formation and cytoadherence ofR jalciparum-infected erythrocytes to vascular endo-thelium result in the sequestration of infected cellsparticularly in the brain vasculature (resulting inanoxia altered brain function and coma) and thusrepresent a major cause of cerebral malaria [65]However the role of the immune system is likely also tobe of critical importance [66] Cytoadherence ofRBCscontaining mature-stage parasites to vascular endo-thelium and platelets is a protective mechanism used bythe parasite to avoid elimination This is mediated byseveral membrane proteins receptors present onendothelial cells (CD36 ICAM-l PECAM-l TSPchondroitin sulfate) and cryptic antigens ofRBCs likethose exposed on altered Band 3 [67] The parasitizedRBCs not only adhere to vascular endothelium but alsoadhere to uninfected RBCs a process known asrosette formation This is mainly mediated by thecomplement receptor CRI (CD35 carrier of Knoppsantigens) since among a large series of null varianterythrocytes tested for the ability to form rosettes onlythose of the Helgenson phenotype (CRI-deficient)were negative In addition Sla( - ) red cells which havea reduced copy number of CRl rosetted poorly [68]The counter receptor of CRI is PfEMP 1 (R jalciparumErythrocyte Membrane Protein 1) a protein of 300-350 kDa produced by a gene of the varfamily but otherligands of PfEMP 1 (heparin sulfate glycans ABOgroups CD36) are also involved in rosette formation[69] The main function of CRI (190-280 kDa) is tocapture and remove from the liver and spleen immune

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complexes containing C3b and C4b but it also plays arole in the immune response [70] The CR1 level onRBCs is low but may vary widely The extramembranous region of CR1 is made of 30 shortconsensus repeats (SCR) and two Knopps blood grouppolymorphisms have been assigned to the homologousregion D within SCR24 (McCab = K1590E) andSCR25 (SlaNil = R160 1G) [71]

Duffy polymorphism and malaria

Malaria is an important selective force for humangenetic adaptations due to the sustained lethal impactthat it has had on human populations around theworld Homozygosity for the Duffy-negative bloodgroup antigen confers complete resistance to vivaxmalaria Nevertheless it is unclear whether selectivepressure of vivax malaria alone was the main cause forthe emergence and fixation of the FYB null FYB null

genotype in much of West Africa where vivax malariais absent Indeed there is little doubt that malariacaused by E vivax is not as directly lethal asE jalciparum but a fulminant E vivax infection stillcauses serious morbidity in those living at theminimum subsistence level The emergence of thenew FYA allele carrying the - 33TC mutation inE vivax-endemic region of Papua New Guineafurther supported the hypothesis that E vivax malariacan act as a selective agent of the Fy(a - b - )phenotypes in this region [7]

Accordingly Fy mRNA and DARC are normallyexpressed in nonerythroid tissues in populations ofWest Mrica where E vivax is no longer present It isassumed that the most common phenotypeFy(a - b - ) represents an adaptive response to resistmalarial infection [325]

RBCs from individuals homozygous for the wild-type promoter (Genotypes FYAFYA FYBFYB andFYAFYB) express twice the amount of Fy antigenthan those heterozygous for the GATA-1 mutation(genotypes FYAFYB SE FYBFYB SE) [727] but thebiological significance of this finding is unknown [7]

In individuals who present the GATA mutation inthe heterozygous form dose effect has been demon-strated being that only 50 of the Duffy antigens areexpressed in the erythrocytes [73] These data suggestthat heterozygosity for the - 33TC mutation in theallele promotes the protection against E vivaxinfection but is still susceptible to it [74]

When we correlated the frequency of - 33TCmutation and the development of malaria by E vivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygotes does not give protection against E vivaxinfection [8]

Another study among patients infected by E vivax inBrazil showed that the proportion of individuals thatdid not present the - 33TC mutation in homozygotes

Duffy bloodgroup and malaria 395

was similar among those infected by E vivax and non-Evivax If it were the case that the mutation inheterozygous form conferred any degree of protectionagainst E vivax infection one would expect toencounter a relative excess of patients without themutation among individuals infected by E vivax [75]

In vitro binding assays have proved a significantdecline in binding activity with heterozygous Duffy-positivenegative genotypes both FYAFYA null andFYBFYBnull

when compared to the homozygousDuffy-positive erythrocytes In these assays cytoad-herence between the DBP ligand domain anderythrocytes from Duffy promoter heterozygousdonors was significantly reduced [74] Previousstudies have confirmed that heterozygous Duffy-negative individuals remain susceptible to infectionsby E vivax [76] studies made by Michon et al [74]suggested that even at the heterozygous state theDuffy-negative allele can confer a quantifiableresistance advantage against E vivax

In all 23 individuals from Papua New Guinea foundwith the FYA null allele all were heterozygous(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withE vivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes from heterozygous individuals carryingthis mutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstE vivax malaria [74]

Other studies demonstrated the presence of RBCswhich react weakly with anti-Fya in eight individuals ofThai ethnicity who live in a region endemic to malaria[77] These phenotypes may occur due to the decreasein the number of RBC antigens to structuralalteration of the antigen itself or even to both motifs[77] This weak Fya antigen could in a manner similarto that observed in Africa offer a selective advantageto this population in relation to malaria as the Fyaantigen is predominant in the region

Among Indonesians who live in regions endemic tomalaria a study was carried out on the phenotypingand genotyping and the presence of the antigen Fyawith low reactivity to the anti-Fya was observed alsooccurring a discrepancy between the genotype andphenotype of these individuals suggesting anothergenetic alternative for the individuals presentingFy(a - b - ) different from the classic genetic basefor this phenotype in Mricans [37]

The presence of the Fya antigen which is weaklyreactive was also described in Malaysians [38]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null

allele Four individuals presented the Fy(a + b - )

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phenotype the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the FYB allele can confer a certain degree ofprotection against infection by Fvivax despite beingdemonstrated by Michon et al [74] a smallerexpression of the Duffy antigens in the RBCs ofindividuals with the - 33TC mutation and thatsituation could be exacerbated by the presence of themutation in both FYA and FYB allele

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

Summary

Since the molecular basis of the Duffy blood grouppolymorphisms has been determined the molecularmechanism that gives rise to the phenotypeFy(a - b - ) in black individuals has been classicallyassociated with a point mutation - 33TC in thepromoter region of a FY B allele a mutation thatwhen present in homozygosity confers protectionagainst Fvivax infection [5]

The described FYA null appears to have a morerecent origin than that of FYB null and this allelehas been reported to occur in New Guinea [7] andBrazil [8]

RBCs from individuals homozygous for the- 33TC mutation express twice the amount of Fyantigen than those heterozygous for the mutation butthe biological significance of this finding is unknown[727]

In individuals who present the GATA mutation inthe heterozygous form dose effect of Duffy antigenshas been demonstrated [25] and these data suggestthat heterozygosity for the - 33TC mutation pro-motes protection against F vivax infection butremains susceptible to it [74]

When we correlated the frequency of the - 33TCmutation and the development of malaria by Fvivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygous individuals does not give protectionagainst Fvivax infection [8]

In individuals from Papua New Guinea found tohave the FYA null allele all of them were heterozygous

(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withFvivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes in heterozygous individuals carrying thismutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstFvivax malaria [74]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null alleleFour individuals presented the Fy(a + b - ) pheno-type the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the mutation in the FYB allele can confer acertain degree of protection against infection byFvivax

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

References

[1] Mollison PL Engelfried CP Contreras M Blood transfusionin clinical medicine Oxford England Blackwell 1997

[2] Collins A Keast BJ Dracopoli N Shields DC Morton NEIntegration of gene maps Chromosome 1 Proc Nat Acad SciUSA 1992894598-4602

[3] Cutbush M Mollison PL Parkin DM A new human bloodgroup Nature 1950165188-189

[4] Sanger R Race RR Jack J The Duffy blood groups of NewYork negroes The phenotype Fy(a-b-) Br J Haematol19551370-374

[5] Tournamille C Colin Y Cartron JP Le Van Kim CDisruption of a GATA motif in the Duffy gene promoterabolishes erythroid gene expression in Duffy-negative individ-uals Nat Genet 199510224-228

[6] Chown B Lewis M Kaita H The Duffy blood group system incaucasians Evidence for a new allele Am J Hum Genet196517384-389

[7] Zimmerman PA Woolley I Masinde GL Miller SMMcNamara DT Hazlett F et al Emergence of FYA null ina Plasmodium vivax-endemic region of Papua New GuineaProc Nat Acad Sci USA 199996 13973-13977

[8] Langhi DM Albuquerque SR Covas DT Perez CA BordinJO The presence ofFYAnull allele of Duffy blood group system

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transmembrane a-helices short protruding hydro-philic loops and an intracellular C-terminal domainof 28 residues [28]

The gp-Fy is composed of the Fya and Fyb epitopesin addition to the Fy3 Fy4 Fy5 and Fy6 epitopesThe Fya e Fyb epitopes are located in the extracellulardomain in resides 42 and the Fy3 epitope is locatedon the third extracellular loop The Fy6 epitope hasbeen accurately mapped to a heptapeptide comprisingresidues 19-25 and is located between the twoglycosylation sites [29]

The Duffy protein is encoded by a short single copygene (15 kb 2 exons) located on chromosome 1 andexhibits significant protein sequence homology withthe human and rabbit IL-8 receptors DARC is mostprobably organized in seven transmembrane domainssimilar to other members of the G-protein coupledchemokine receptors [30] The major (336 aa) andminor (338 aa) DARC polypeptide isoforms areencoded by the spliced and unspliced Fy mRNAsrespectively which differ by the sequence of the sixand eight NHTterminal amino acids respectively butbind anti-Fy antibodies and chemokines equally wellDARC has a specialized tissue distribution as presenton RBCs endothelial cells of post-capillary venules(a site of leukocyte trafficking) Purkinje cells of thecerebellum and presumably on some epithelial cells inthe kidney [3132]

Duffy polymorphism

The molecular basis of the Duffy blood grouppolymorphisms has been determined The Duffy(FY) gene is located in the 1q22-q23 region ofchromosome 1 [33] The Duffy cDNA was originallycloned from a non-spliced mRNA and it was assumedthat Duffy is an intron less gene [34] The Duffytranscription unit encompasses 1572 nt includingexon 1 of 55 nt a single intron of 479 nt and exon 2 of1038 nt [35]

Duffy blood group and malaria 391

The Fyab antigenic polymorphism results from apoint mutation at nucleotide 125 in the gp-FY genewith the change of a single base G 125A This changeresults in a single amino acid difference changingglycine by aspartic acid at position 42 of gp-FyGlycine at position 42 (Gly42) defines the Fya antigenand aspartic acid at position 42 (Asp42) defines theFyb antigen [34] (Figure 1)

The molecular mechanism that gives rise to thephenotype Fy(a - b - ) in black individuals has beenclassically associated with a point mutation - 33TC inthe promoter region of a FY B allele that disrupts abinding site for the transcription factor GATA-1 amutation that specifically abolishes the transcriptionalactivity in erythroid cells GATA box of individuals thatexpress this mutation in homozygosity [5] (Figure 2)This mutation defines the FYB SE allele that isstructurally identical to the FYB allele in its codifyingregion

Individuals showing heterozygosity for the GATAbox mutation demonstrate dosage effect and haveapproximately one-half the level of Duffy antigen inerythrocytes [25]

In Caucasians the Fy(a - b -) phenotype isextremely rare and in all cases studied so far it isassociated with mutations or deletions within thecoding sequence of the DARe gene [32] It is assumedthat these mutations in contrast to the erythroidspecific mutation - 33TC should lead to the trueF~ull phenotype with a lack of expression ofDARC inboth erythroid and nonerythroid cells

For individuals from malarial endemic regions insoutheastern Asia the Fy(a - b -) phenotype ispractically nonexistent and the molecular basis of thisphenotype is not the same as that described in Blackindividuals in other words homozygosity for theFYB ES allele [37] In some locations individualshaving the Fy(a - b - ) phenotype possess the FYA-FYA and FYAFYB genotypes without the presence ofthe FYBESFYBES genotype [37] therefore there isheterogeneity in the findings In this group of

Duffy 5Gene

mRNA

Exon 1

------

Promoter region -

5

Exon 2 1572 (nts)

3

gp-Fy NHZ COOHbull42 (residue number)

Fya Gly

Fyb Asp

Figure 1 Schematic representations of the Duffy gene FY (top) mRNA (middle) and gp-Fy (bottom) with polymorphism FYAFYB at nt125 and residues Gly for FYA and Asp for FYB at position 42 (Modified from Pogo Chaudhuri [32])

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392 D M Langhi amp J O Bordin

Point mutationnt-33T -7 C

FYA or FYB Gene

FYBSE Gene

I J

GATA-l

Coding Region 1008

3

Figure 2 Schematic representation of point mutation in GATA-I site responsible for the non-expression of gp-Fy in RBCs of blackindividuals with Fy(a - b - ) phenotype of the mutation point at the bonding site of GAT A-I not the expression of the GPD in RBCs of themajority of Black individuals with the Fy(a - b - ) phenotype (Modified from Issit Anstee [36])

individuals a study was carried out on the phenotypingand genotyping and the presence of the antigen Fyawkwith low reactivity to anti-Fya was observed

The presence of the Fya antigen which is weaklyreactive was also described in Malaysians [38]

Recently Zimmerman et al [7] described thepresence of this mutation linked to the FYA allele in apopulation living in a malaria endemic region of PapuaNew Guinea and this FYA null appears to have a morerecent origin than that of FYB null

We demonstrated the presence of the FYA null allelein blood donors and Individuals from a malarialendemic region of Brazil [8]

The Fybwkphenotype is associated with a missensemutation in the coding region of FYB gene with asingle substitution at nt 265C- T that produces anamino acid change Arg89Cys in gp-Fy Near thismutation another mutation G298A resulting inAlalOOThr amino acid substitution was also ident-ified This mutation is silent and is also present in FYBor FYA alleles from Fy(a - b +) and Fy(a + b + )individuals [25] These amino acid substitutions occurin the first intracellular loop of gp-Fy and only theArg89Cys substitution produces the Fy(a - b + wk)phenotype (Figure 3)

The amino acid substitutions occur in the firstintracellular loop of gp-Fy representing considerable

modification in the chemical nature of the site andresults in very low membrane expression of DARC inFy(a - bweak)erythrocytes entailing a weakening ofthe Fyb antigen [34]

The expression of Fy3 Fy6 and Fyb antigens whenthe G298A mutation is present is similar to thoseencoded by cDNA of FYB contrary to the situationwhen the mutation C265T exists [25]

In non-Ashkenazi Jews individuals with thephenotype Fy(a - b -) show the promoter wild-type GATA however Non-Ashkenazi Jewish indivi-duals with the Fy(a - b -) phenotype weredescribed as presenting the region which promotesthe FYB gene in wild type form but with the C265Tmutation in the codifying region of the FY gene whichalters the antigenic determinants of the DARCweakening the Fyb antigen [39]

Also described were non-Ashkenazi Jewish indivi-duals with the Fy(a - b -) phenotype and theFYBFYB genotype heterozygotes for the GATAmutation entailing simultaneously the C265Tmutation weakener of FYB [39]

Similar to that which was described in non-Ashkenazi Jews was also described in BrazilianBlack individuals namely the association of themutation responsible for the FYB fraco genotype(C265T) with the T-33C mutation (FYB es) in the

Duffy 5Gene

mRNA

Exon 1

------ Promoter 1

region bullbull I

5

Exon 2

1008 (nts)

bullbullbull-3AAAn

gp-Fy NH2 bullbull bullbull89 100 (residue number)

ArgCys Alaffhr

Figure 3 Schematic representation of FY gene with the C265T mutation (FYB WK) encoding the amino acid change Arg89Cys and thesilent mutation G298A codifying the amino acid change AlalOOThr (Modified from Pogo Chaudhuri [32])

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individuals whose Fy(b -) phenotype cannot beexplained by the isolated mutation in the GATA-1box [40]

Functional aspects

Although all blood group antigens are serologicallydetectable on RBCs most of them are also expressedin non-erythroid tissues raising further questions ontheir physiological function under normal andpathological conditions In addition to their structuraldiversity blood group antigens also possess widefunctional diversity and can be schematically sub-divided into five classes (a) transporters and channels(b) receptors for ligands viruses bacteria andparasites (c) adhesion molecules (d) enzymes and(e) structural proteins

Many RBCs surface molecules among those whichcarry blood groups are receptors for viruses bacteriaand parasites suggesting that these antigens may playadirect role in the pathogenesis of infectious diseases[4142]

A variety of microorganisms recognize carbohydratestructures present on glycolipids and glycoproteinsfor instance sialic acids which are abundantlyrepresented on glycophorin A (GPA) or the Cala1-4 Gal motif shared by P Pk and PI glycolipids usedby several bacterial strains and toxins responsible forupper urinary tract infections [43]

The Duffy antigens (Fyab Fy3 and Fy6) are carriedby a membrane glycoprotein exhibiting two interestingbiological properties

bull as a promiscuous receptor for chemokines of theCC (RANTES MCP-1) and CXC (IL8 mgSA)subfamilies of proinflammatory peptides namedaccording to the structure of a conserved cysteine(C) motif Hence the Duffy protein was renamedDARC for Duffy AntigenReceptor for Chemo-kines

bull as erythroid receptor for R vivax and R knowlesi

Duffy antigen receptor for chemokine

The fact that RBCs possess a chemokine receptor wasfirst established by Darbone et al [44]

Chemokines constitute a family of proinflammatorycytokines capable of activating leukocytes and causingchemotaxis but other important functions have beendiscovered including angiogenic and angiostaticactivities [45] The number and spacing of amino-terminal cysteines have been used in the classificationof chemokines into four families C CC CXC andCXXXC The biological effect of chemokines ismediated by the binding and activation of G-protein-coupled seven-transmembrane domain chemokinereceptors [46] Some chemokines (classes CC and

Duffy blood group and malaria 393

CXC) also bind to the Duffy blood group glyco-protein which is now called DARC

Of the cellular chemokine receptors CXCR4CCR5 and Duffy are the only receptors that havebeen unequivocally proven to act as coreceptors forcell entry of pathogens Duffy for the entry of malarialparasite R vivax and CCR5 for the entry of M-tropicstrains of human immunodeficiency virus (HIV) [47]

During experiments with chemokine receptors inerythrocytes the perfect correlation between thebonding of the chemokines to the erythrocytes andthe presence of the Duffy antigen was well established[12] It has been demonstrated that anti-Fy antibodiesinhibited the chemokine binding ability to Duffypositive erythrocytes and chemokines that bind toerythrocytes block RBC invasion by malarial parasitesthat use the Duffy antigen as a receptor [12]

CC and CXC chemokines bind to DARC with highaffinity suggesting some role in inflammatory reactions[31] DARC might play a role as a scavenger on theRBC surface to eliminate excess of toxic chemokinesproduced in some pathologic situations [44]

There are some experimental studies suggestingthat DARC is a redundant protein that may playa rolein the regulation of induced leukocyte trafficking invivo [48]

The CXCR4 and CCR5 chemokine receptors aremandatory cofactors for HIV infection by interactingwith the viral envelope gp 120 in the presence of CD4[49] A report shows that HIV-1 viral particles mightbind to the Fy protein suggesting that RBCs mayfunction as a virus reservoir or as a receptor for theentry in some cells [50]

Duffy antigen receptor for R vivax

R vivax causes approximately between 70 and 80million cases of malaria per year and is the most amplydistributed human malaria in the world [51]

One of the most interesting aspects of the Duffyantigen is its function as a receptor for the humanmalaria parasite R vivax

In 1975 Miller et al [9] showed that Duffy-negative human RBCs were resistant to invasion byR knowlesi a monkey malaria parasite that was knownto be capable of invading human RBCs and in rareinstances of infecting humans Furthermore anti-Fyaand anti-Fyb blocked invasion of R knowlesi intoFy(a + b -) and Fy(a - b +) RBCs respectivelyand the treatment of the RBCs with enzymes whichremove from their surface the antigenic determinantsFya e Fyb (chemotrypsin and pronase) making theRBCs resistant to invasion [9]

Studies on R knowlesi were extended to R vivax ahuman malaria that is second only to Plasmodiumfaleiparum in terms of the toll it takes on populationsof endemic areas Although R vivax is widespreadthroughout the tropical and subtropical world and

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394 D M Langhi amp J O Bordin

is a major drain on the health care resources of Indiaand Southeast Asia it is absent from West Africawhere more than 95 of the population is Duffynegative Miller et al [52] showed that resistance toR vivax correlates with the Duffy-negative pheno-type and more recently Barnwell et al [53] showedthat R vivax merozoites are incapable of invadingDuffy-negative RBCs Thus R vivax like R knowlesirelies on a Duffy antigen-parasite ligand interactionsfor invasion It should be noted that R vivaxpreferentially invades reticulocytes [54] althoughmature RBCs as well as reticulocytes express acoreceptor that along with the Duffy antigen isnecessary for optimal invasion by R vivax Thebonding agent of the R vivax parasite whichspecifically bonds itself to the reticulocytes was alsoidentified and the cDNA encoding this protein wascloned [54]

When the anti-Fy6 antibody became availablestudies demonstrated that the presence of Fy6 is whatresults from the invasion of human RBCs by themerozoites of the R vivax [53]

In humans the presence or absence of Fy6correlates with the presence or absence of the Fyaand Fyb In other words the Fy(a +) or Fy(b + )RBCs are always Fy6 positive and Fy(a - b - ) RBCsare Fy6 negative The invasion of the Fy(a + ) RBCsby R vivax can be partially blocked by the covering ofthe RBC with anti-Fya [9] and totally blocked bycovering the RBC with anti-Fy6 [55]

The fact that the great majority of black African andAfro-American individuals are resistant to infection byR vivax is directly related to the Fy(a - b - )phenotype or in other words the absence of the Fyaand Fyb antigens

One Duffy binding protein of R vivax calledPvDAP-l (Pv - indicates R vivax and DAPindicates Duffy-associating protein) has a criticalrole as a bonder in the adhesion of the parasite andposterior invasion of the positive Duffy RBCs [56] Rvivax as with other species of human Plasmodiuminitiate erythrocyte invasion through the expression ofvarious surface organelles and structures on themerozoite which bind with the surface proteins ofthe erythrocyte The well-characterized interactionof the bonder-receptor involves the Duffy bondingprotein expressed in the merozoite form of theR vivax and its corresponding receptor in theerythrocyte the receptor DARC This interaction isunique among human malaria infections where theinteraction of the bonding receptor is essential to theinvasion of the erythrocyte by R vivax Alternatemodels of erythrocytic invasion have not yet beendescribed [57] This Duffy bonding protein ofR vivax belongs to the family of bonding proteinsfor erythrocytes which also includes the bondingprotein for sialic acid R jalciparum and to the Duffybonding protein R knowlesi

R vivax merozoites invade reticulocytes that expressthe Duffy protein and the counter receptor is a140 kDa protein (PvDBP) that belongs to Plasmodiumadhesion proteins including EBA-175 and PfEMPl(see above) all having in the NHz-ter region acysteine-rich domain (called DPP region II) mediat-ing erythrocyte binding [5859]

It is not known whether the Duffy antigen is astructural junction component because the molecularnature of junction has yet to be determined It is clearhowever that the antigen interaction of the parasiteDuffy-receptor is crucial to the formation of thejunction and subsequent invasion [57]

Several blood group molecules of red cell surfacecontribute to the complex mechanisms of invasion andsequestration steps that characterize the most severeform of malaria caused by R jalciparum R jalciparummerozoites may invade RBCs through sialic acid-dependent and independent pathways [6061] GPA isinvolved in the sialic-acid dependent pathway and thecounter receptor of GPA is the parasite proteinerythrocyte binding antigen (EBA) of 175 kDa [62]which binds to sialic acid and peptide backbone ofGPAthrough a cysteine-rich domain (region II) analogousto the one mediating R vivaxDuffy protein interaction[63] Recently a homologue to EBA-175 calledBAEBL has been shown to use GPC for invasion[64] Rosette formation and cytoadherence ofR jalciparum-infected erythrocytes to vascular endo-thelium result in the sequestration of infected cellsparticularly in the brain vasculature (resulting inanoxia altered brain function and coma) and thusrepresent a major cause of cerebral malaria [65]However the role of the immune system is likely also tobe of critical importance [66] Cytoadherence ofRBCscontaining mature-stage parasites to vascular endo-thelium and platelets is a protective mechanism used bythe parasite to avoid elimination This is mediated byseveral membrane proteins receptors present onendothelial cells (CD36 ICAM-l PECAM-l TSPchondroitin sulfate) and cryptic antigens ofRBCs likethose exposed on altered Band 3 [67] The parasitizedRBCs not only adhere to vascular endothelium but alsoadhere to uninfected RBCs a process known asrosette formation This is mainly mediated by thecomplement receptor CRI (CD35 carrier of Knoppsantigens) since among a large series of null varianterythrocytes tested for the ability to form rosettes onlythose of the Helgenson phenotype (CRI-deficient)were negative In addition Sla( - ) red cells which havea reduced copy number of CRl rosetted poorly [68]The counter receptor of CRI is PfEMP 1 (R jalciparumErythrocyte Membrane Protein 1) a protein of 300-350 kDa produced by a gene of the varfamily but otherligands of PfEMP 1 (heparin sulfate glycans ABOgroups CD36) are also involved in rosette formation[69] The main function of CRI (190-280 kDa) is tocapture and remove from the liver and spleen immune

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complexes containing C3b and C4b but it also plays arole in the immune response [70] The CR1 level onRBCs is low but may vary widely The extramembranous region of CR1 is made of 30 shortconsensus repeats (SCR) and two Knopps blood grouppolymorphisms have been assigned to the homologousregion D within SCR24 (McCab = K1590E) andSCR25 (SlaNil = R160 1G) [71]

Duffy polymorphism and malaria

Malaria is an important selective force for humangenetic adaptations due to the sustained lethal impactthat it has had on human populations around theworld Homozygosity for the Duffy-negative bloodgroup antigen confers complete resistance to vivaxmalaria Nevertheless it is unclear whether selectivepressure of vivax malaria alone was the main cause forthe emergence and fixation of the FYB null FYB null

genotype in much of West Africa where vivax malariais absent Indeed there is little doubt that malariacaused by E vivax is not as directly lethal asE jalciparum but a fulminant E vivax infection stillcauses serious morbidity in those living at theminimum subsistence level The emergence of thenew FYA allele carrying the - 33TC mutation inE vivax-endemic region of Papua New Guineafurther supported the hypothesis that E vivax malariacan act as a selective agent of the Fy(a - b - )phenotypes in this region [7]

Accordingly Fy mRNA and DARC are normallyexpressed in nonerythroid tissues in populations ofWest Mrica where E vivax is no longer present It isassumed that the most common phenotypeFy(a - b - ) represents an adaptive response to resistmalarial infection [325]

RBCs from individuals homozygous for the wild-type promoter (Genotypes FYAFYA FYBFYB andFYAFYB) express twice the amount of Fy antigenthan those heterozygous for the GATA-1 mutation(genotypes FYAFYB SE FYBFYB SE) [727] but thebiological significance of this finding is unknown [7]

In individuals who present the GATA mutation inthe heterozygous form dose effect has been demon-strated being that only 50 of the Duffy antigens areexpressed in the erythrocytes [73] These data suggestthat heterozygosity for the - 33TC mutation in theallele promotes the protection against E vivaxinfection but is still susceptible to it [74]

When we correlated the frequency of - 33TCmutation and the development of malaria by E vivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygotes does not give protection against E vivaxinfection [8]

Another study among patients infected by E vivax inBrazil showed that the proportion of individuals thatdid not present the - 33TC mutation in homozygotes

Duffy bloodgroup and malaria 395

was similar among those infected by E vivax and non-Evivax If it were the case that the mutation inheterozygous form conferred any degree of protectionagainst E vivax infection one would expect toencounter a relative excess of patients without themutation among individuals infected by E vivax [75]

In vitro binding assays have proved a significantdecline in binding activity with heterozygous Duffy-positivenegative genotypes both FYAFYA null andFYBFYBnull

when compared to the homozygousDuffy-positive erythrocytes In these assays cytoad-herence between the DBP ligand domain anderythrocytes from Duffy promoter heterozygousdonors was significantly reduced [74] Previousstudies have confirmed that heterozygous Duffy-negative individuals remain susceptible to infectionsby E vivax [76] studies made by Michon et al [74]suggested that even at the heterozygous state theDuffy-negative allele can confer a quantifiableresistance advantage against E vivax

In all 23 individuals from Papua New Guinea foundwith the FYA null allele all were heterozygous(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withE vivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes from heterozygous individuals carryingthis mutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstE vivax malaria [74]

Other studies demonstrated the presence of RBCswhich react weakly with anti-Fya in eight individuals ofThai ethnicity who live in a region endemic to malaria[77] These phenotypes may occur due to the decreasein the number of RBC antigens to structuralalteration of the antigen itself or even to both motifs[77] This weak Fya antigen could in a manner similarto that observed in Africa offer a selective advantageto this population in relation to malaria as the Fyaantigen is predominant in the region

Among Indonesians who live in regions endemic tomalaria a study was carried out on the phenotypingand genotyping and the presence of the antigen Fyawith low reactivity to the anti-Fya was observed alsooccurring a discrepancy between the genotype andphenotype of these individuals suggesting anothergenetic alternative for the individuals presentingFy(a - b - ) different from the classic genetic basefor this phenotype in Mricans [37]

The presence of the Fya antigen which is weaklyreactive was also described in Malaysians [38]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null

allele Four individuals presented the Fy(a + b - )

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396 D M Langhi ampJ 0 Bardin

phenotype the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the FYB allele can confer a certain degree ofprotection against infection by Fvivax despite beingdemonstrated by Michon et al [74] a smallerexpression of the Duffy antigens in the RBCs ofindividuals with the - 33TC mutation and thatsituation could be exacerbated by the presence of themutation in both FYA and FYB allele

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

Summary

Since the molecular basis of the Duffy blood grouppolymorphisms has been determined the molecularmechanism that gives rise to the phenotypeFy(a - b - ) in black individuals has been classicallyassociated with a point mutation - 33TC in thepromoter region of a FY B allele a mutation thatwhen present in homozygosity confers protectionagainst Fvivax infection [5]

The described FYA null appears to have a morerecent origin than that of FYB null and this allelehas been reported to occur in New Guinea [7] andBrazil [8]

RBCs from individuals homozygous for the- 33TC mutation express twice the amount of Fyantigen than those heterozygous for the mutation butthe biological significance of this finding is unknown[727]

In individuals who present the GATA mutation inthe heterozygous form dose effect of Duffy antigenshas been demonstrated [25] and these data suggestthat heterozygosity for the - 33TC mutation pro-motes protection against F vivax infection butremains susceptible to it [74]

When we correlated the frequency of the - 33TCmutation and the development of malaria by Fvivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygous individuals does not give protectionagainst Fvivax infection [8]

In individuals from Papua New Guinea found tohave the FYA null allele all of them were heterozygous

(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withFvivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes in heterozygous individuals carrying thismutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstFvivax malaria [74]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null alleleFour individuals presented the Fy(a + b - ) pheno-type the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the mutation in the FYB allele can confer acertain degree of protection against infection byFvivax

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

References

[1] Mollison PL Engelfried CP Contreras M Blood transfusionin clinical medicine Oxford England Blackwell 1997

[2] Collins A Keast BJ Dracopoli N Shields DC Morton NEIntegration of gene maps Chromosome 1 Proc Nat Acad SciUSA 1992894598-4602

[3] Cutbush M Mollison PL Parkin DM A new human bloodgroup Nature 1950165188-189

[4] Sanger R Race RR Jack J The Duffy blood groups of NewYork negroes The phenotype Fy(a-b-) Br J Haematol19551370-374

[5] Tournamille C Colin Y Cartron JP Le Van Kim CDisruption of a GATA motif in the Duffy gene promoterabolishes erythroid gene expression in Duffy-negative individ-uals Nat Genet 199510224-228

[6] Chown B Lewis M Kaita H The Duffy blood group system incaucasians Evidence for a new allele Am J Hum Genet196517384-389

[7] Zimmerman PA Woolley I Masinde GL Miller SMMcNamara DT Hazlett F et al Emergence of FYA null ina Plasmodium vivax-endemic region of Papua New GuineaProc Nat Acad Sci USA 199996 13973-13977

[8] Langhi DM Albuquerque SR Covas DT Perez CA BordinJO The presence ofFYAnull allele of Duffy blood group system

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Point mutationnt-33T -7 C

FYA or FYB Gene

FYBSE Gene

I J

GATA-l

Coding Region 1008

3

Figure 2 Schematic representation of point mutation in GATA-I site responsible for the non-expression of gp-Fy in RBCs of blackindividuals with Fy(a - b - ) phenotype of the mutation point at the bonding site of GAT A-I not the expression of the GPD in RBCs of themajority of Black individuals with the Fy(a - b - ) phenotype (Modified from Issit Anstee [36])

individuals a study was carried out on the phenotypingand genotyping and the presence of the antigen Fyawkwith low reactivity to anti-Fya was observed

The presence of the Fya antigen which is weaklyreactive was also described in Malaysians [38]

Recently Zimmerman et al [7] described thepresence of this mutation linked to the FYA allele in apopulation living in a malaria endemic region of PapuaNew Guinea and this FYA null appears to have a morerecent origin than that of FYB null

We demonstrated the presence of the FYA null allelein blood donors and Individuals from a malarialendemic region of Brazil [8]

The Fybwkphenotype is associated with a missensemutation in the coding region of FYB gene with asingle substitution at nt 265C- T that produces anamino acid change Arg89Cys in gp-Fy Near thismutation another mutation G298A resulting inAlalOOThr amino acid substitution was also ident-ified This mutation is silent and is also present in FYBor FYA alleles from Fy(a - b +) and Fy(a + b + )individuals [25] These amino acid substitutions occurin the first intracellular loop of gp-Fy and only theArg89Cys substitution produces the Fy(a - b + wk)phenotype (Figure 3)

The amino acid substitutions occur in the firstintracellular loop of gp-Fy representing considerable

modification in the chemical nature of the site andresults in very low membrane expression of DARC inFy(a - bweak)erythrocytes entailing a weakening ofthe Fyb antigen [34]

The expression of Fy3 Fy6 and Fyb antigens whenthe G298A mutation is present is similar to thoseencoded by cDNA of FYB contrary to the situationwhen the mutation C265T exists [25]

In non-Ashkenazi Jews individuals with thephenotype Fy(a - b -) show the promoter wild-type GATA however Non-Ashkenazi Jewish indivi-duals with the Fy(a - b -) phenotype weredescribed as presenting the region which promotesthe FYB gene in wild type form but with the C265Tmutation in the codifying region of the FY gene whichalters the antigenic determinants of the DARCweakening the Fyb antigen [39]

Also described were non-Ashkenazi Jewish indivi-duals with the Fy(a - b -) phenotype and theFYBFYB genotype heterozygotes for the GATAmutation entailing simultaneously the C265Tmutation weakener of FYB [39]

Similar to that which was described in non-Ashkenazi Jews was also described in BrazilianBlack individuals namely the association of themutation responsible for the FYB fraco genotype(C265T) with the T-33C mutation (FYB es) in the

Duffy 5Gene

mRNA

Exon 1

------ Promoter 1

region bullbull I

5

Exon 2

1008 (nts)

bullbullbull-3AAAn

gp-Fy NH2 bullbull bullbull89 100 (residue number)

ArgCys Alaffhr

Figure 3 Schematic representation of FY gene with the C265T mutation (FYB WK) encoding the amino acid change Arg89Cys and thesilent mutation G298A codifying the amino acid change AlalOOThr (Modified from Pogo Chaudhuri [32])

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individuals whose Fy(b -) phenotype cannot beexplained by the isolated mutation in the GATA-1box [40]

Functional aspects

Although all blood group antigens are serologicallydetectable on RBCs most of them are also expressedin non-erythroid tissues raising further questions ontheir physiological function under normal andpathological conditions In addition to their structuraldiversity blood group antigens also possess widefunctional diversity and can be schematically sub-divided into five classes (a) transporters and channels(b) receptors for ligands viruses bacteria andparasites (c) adhesion molecules (d) enzymes and(e) structural proteins

Many RBCs surface molecules among those whichcarry blood groups are receptors for viruses bacteriaand parasites suggesting that these antigens may playadirect role in the pathogenesis of infectious diseases[4142]

A variety of microorganisms recognize carbohydratestructures present on glycolipids and glycoproteinsfor instance sialic acids which are abundantlyrepresented on glycophorin A (GPA) or the Cala1-4 Gal motif shared by P Pk and PI glycolipids usedby several bacterial strains and toxins responsible forupper urinary tract infections [43]

The Duffy antigens (Fyab Fy3 and Fy6) are carriedby a membrane glycoprotein exhibiting two interestingbiological properties

bull as a promiscuous receptor for chemokines of theCC (RANTES MCP-1) and CXC (IL8 mgSA)subfamilies of proinflammatory peptides namedaccording to the structure of a conserved cysteine(C) motif Hence the Duffy protein was renamedDARC for Duffy AntigenReceptor for Chemo-kines

bull as erythroid receptor for R vivax and R knowlesi

Duffy antigen receptor for chemokine

The fact that RBCs possess a chemokine receptor wasfirst established by Darbone et al [44]

Chemokines constitute a family of proinflammatorycytokines capable of activating leukocytes and causingchemotaxis but other important functions have beendiscovered including angiogenic and angiostaticactivities [45] The number and spacing of amino-terminal cysteines have been used in the classificationof chemokines into four families C CC CXC andCXXXC The biological effect of chemokines ismediated by the binding and activation of G-protein-coupled seven-transmembrane domain chemokinereceptors [46] Some chemokines (classes CC and

Duffy blood group and malaria 393

CXC) also bind to the Duffy blood group glyco-protein which is now called DARC

Of the cellular chemokine receptors CXCR4CCR5 and Duffy are the only receptors that havebeen unequivocally proven to act as coreceptors forcell entry of pathogens Duffy for the entry of malarialparasite R vivax and CCR5 for the entry of M-tropicstrains of human immunodeficiency virus (HIV) [47]

During experiments with chemokine receptors inerythrocytes the perfect correlation between thebonding of the chemokines to the erythrocytes andthe presence of the Duffy antigen was well established[12] It has been demonstrated that anti-Fy antibodiesinhibited the chemokine binding ability to Duffypositive erythrocytes and chemokines that bind toerythrocytes block RBC invasion by malarial parasitesthat use the Duffy antigen as a receptor [12]

CC and CXC chemokines bind to DARC with highaffinity suggesting some role in inflammatory reactions[31] DARC might play a role as a scavenger on theRBC surface to eliminate excess of toxic chemokinesproduced in some pathologic situations [44]

There are some experimental studies suggestingthat DARC is a redundant protein that may playa rolein the regulation of induced leukocyte trafficking invivo [48]

The CXCR4 and CCR5 chemokine receptors aremandatory cofactors for HIV infection by interactingwith the viral envelope gp 120 in the presence of CD4[49] A report shows that HIV-1 viral particles mightbind to the Fy protein suggesting that RBCs mayfunction as a virus reservoir or as a receptor for theentry in some cells [50]

Duffy antigen receptor for R vivax

R vivax causes approximately between 70 and 80million cases of malaria per year and is the most amplydistributed human malaria in the world [51]

One of the most interesting aspects of the Duffyantigen is its function as a receptor for the humanmalaria parasite R vivax

In 1975 Miller et al [9] showed that Duffy-negative human RBCs were resistant to invasion byR knowlesi a monkey malaria parasite that was knownto be capable of invading human RBCs and in rareinstances of infecting humans Furthermore anti-Fyaand anti-Fyb blocked invasion of R knowlesi intoFy(a + b -) and Fy(a - b +) RBCs respectivelyand the treatment of the RBCs with enzymes whichremove from their surface the antigenic determinantsFya e Fyb (chemotrypsin and pronase) making theRBCs resistant to invasion [9]

Studies on R knowlesi were extended to R vivax ahuman malaria that is second only to Plasmodiumfaleiparum in terms of the toll it takes on populationsof endemic areas Although R vivax is widespreadthroughout the tropical and subtropical world and

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394 D M Langhi amp J O Bordin

is a major drain on the health care resources of Indiaand Southeast Asia it is absent from West Africawhere more than 95 of the population is Duffynegative Miller et al [52] showed that resistance toR vivax correlates with the Duffy-negative pheno-type and more recently Barnwell et al [53] showedthat R vivax merozoites are incapable of invadingDuffy-negative RBCs Thus R vivax like R knowlesirelies on a Duffy antigen-parasite ligand interactionsfor invasion It should be noted that R vivaxpreferentially invades reticulocytes [54] althoughmature RBCs as well as reticulocytes express acoreceptor that along with the Duffy antigen isnecessary for optimal invasion by R vivax Thebonding agent of the R vivax parasite whichspecifically bonds itself to the reticulocytes was alsoidentified and the cDNA encoding this protein wascloned [54]

When the anti-Fy6 antibody became availablestudies demonstrated that the presence of Fy6 is whatresults from the invasion of human RBCs by themerozoites of the R vivax [53]

In humans the presence or absence of Fy6correlates with the presence or absence of the Fyaand Fyb In other words the Fy(a +) or Fy(b + )RBCs are always Fy6 positive and Fy(a - b - ) RBCsare Fy6 negative The invasion of the Fy(a + ) RBCsby R vivax can be partially blocked by the covering ofthe RBC with anti-Fya [9] and totally blocked bycovering the RBC with anti-Fy6 [55]

The fact that the great majority of black African andAfro-American individuals are resistant to infection byR vivax is directly related to the Fy(a - b - )phenotype or in other words the absence of the Fyaand Fyb antigens

One Duffy binding protein of R vivax calledPvDAP-l (Pv - indicates R vivax and DAPindicates Duffy-associating protein) has a criticalrole as a bonder in the adhesion of the parasite andposterior invasion of the positive Duffy RBCs [56] Rvivax as with other species of human Plasmodiuminitiate erythrocyte invasion through the expression ofvarious surface organelles and structures on themerozoite which bind with the surface proteins ofthe erythrocyte The well-characterized interactionof the bonder-receptor involves the Duffy bondingprotein expressed in the merozoite form of theR vivax and its corresponding receptor in theerythrocyte the receptor DARC This interaction isunique among human malaria infections where theinteraction of the bonding receptor is essential to theinvasion of the erythrocyte by R vivax Alternatemodels of erythrocytic invasion have not yet beendescribed [57] This Duffy bonding protein ofR vivax belongs to the family of bonding proteinsfor erythrocytes which also includes the bondingprotein for sialic acid R jalciparum and to the Duffybonding protein R knowlesi

R vivax merozoites invade reticulocytes that expressthe Duffy protein and the counter receptor is a140 kDa protein (PvDBP) that belongs to Plasmodiumadhesion proteins including EBA-175 and PfEMPl(see above) all having in the NHz-ter region acysteine-rich domain (called DPP region II) mediat-ing erythrocyte binding [5859]

It is not known whether the Duffy antigen is astructural junction component because the molecularnature of junction has yet to be determined It is clearhowever that the antigen interaction of the parasiteDuffy-receptor is crucial to the formation of thejunction and subsequent invasion [57]

Several blood group molecules of red cell surfacecontribute to the complex mechanisms of invasion andsequestration steps that characterize the most severeform of malaria caused by R jalciparum R jalciparummerozoites may invade RBCs through sialic acid-dependent and independent pathways [6061] GPA isinvolved in the sialic-acid dependent pathway and thecounter receptor of GPA is the parasite proteinerythrocyte binding antigen (EBA) of 175 kDa [62]which binds to sialic acid and peptide backbone ofGPAthrough a cysteine-rich domain (region II) analogousto the one mediating R vivaxDuffy protein interaction[63] Recently a homologue to EBA-175 calledBAEBL has been shown to use GPC for invasion[64] Rosette formation and cytoadherence ofR jalciparum-infected erythrocytes to vascular endo-thelium result in the sequestration of infected cellsparticularly in the brain vasculature (resulting inanoxia altered brain function and coma) and thusrepresent a major cause of cerebral malaria [65]However the role of the immune system is likely also tobe of critical importance [66] Cytoadherence ofRBCscontaining mature-stage parasites to vascular endo-thelium and platelets is a protective mechanism used bythe parasite to avoid elimination This is mediated byseveral membrane proteins receptors present onendothelial cells (CD36 ICAM-l PECAM-l TSPchondroitin sulfate) and cryptic antigens ofRBCs likethose exposed on altered Band 3 [67] The parasitizedRBCs not only adhere to vascular endothelium but alsoadhere to uninfected RBCs a process known asrosette formation This is mainly mediated by thecomplement receptor CRI (CD35 carrier of Knoppsantigens) since among a large series of null varianterythrocytes tested for the ability to form rosettes onlythose of the Helgenson phenotype (CRI-deficient)were negative In addition Sla( - ) red cells which havea reduced copy number of CRl rosetted poorly [68]The counter receptor of CRI is PfEMP 1 (R jalciparumErythrocyte Membrane Protein 1) a protein of 300-350 kDa produced by a gene of the varfamily but otherligands of PfEMP 1 (heparin sulfate glycans ABOgroups CD36) are also involved in rosette formation[69] The main function of CRI (190-280 kDa) is tocapture and remove from the liver and spleen immune

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complexes containing C3b and C4b but it also plays arole in the immune response [70] The CR1 level onRBCs is low but may vary widely The extramembranous region of CR1 is made of 30 shortconsensus repeats (SCR) and two Knopps blood grouppolymorphisms have been assigned to the homologousregion D within SCR24 (McCab = K1590E) andSCR25 (SlaNil = R160 1G) [71]

Duffy polymorphism and malaria

Malaria is an important selective force for humangenetic adaptations due to the sustained lethal impactthat it has had on human populations around theworld Homozygosity for the Duffy-negative bloodgroup antigen confers complete resistance to vivaxmalaria Nevertheless it is unclear whether selectivepressure of vivax malaria alone was the main cause forthe emergence and fixation of the FYB null FYB null

genotype in much of West Africa where vivax malariais absent Indeed there is little doubt that malariacaused by E vivax is not as directly lethal asE jalciparum but a fulminant E vivax infection stillcauses serious morbidity in those living at theminimum subsistence level The emergence of thenew FYA allele carrying the - 33TC mutation inE vivax-endemic region of Papua New Guineafurther supported the hypothesis that E vivax malariacan act as a selective agent of the Fy(a - b - )phenotypes in this region [7]

Accordingly Fy mRNA and DARC are normallyexpressed in nonerythroid tissues in populations ofWest Mrica where E vivax is no longer present It isassumed that the most common phenotypeFy(a - b - ) represents an adaptive response to resistmalarial infection [325]

RBCs from individuals homozygous for the wild-type promoter (Genotypes FYAFYA FYBFYB andFYAFYB) express twice the amount of Fy antigenthan those heterozygous for the GATA-1 mutation(genotypes FYAFYB SE FYBFYB SE) [727] but thebiological significance of this finding is unknown [7]

In individuals who present the GATA mutation inthe heterozygous form dose effect has been demon-strated being that only 50 of the Duffy antigens areexpressed in the erythrocytes [73] These data suggestthat heterozygosity for the - 33TC mutation in theallele promotes the protection against E vivaxinfection but is still susceptible to it [74]

When we correlated the frequency of - 33TCmutation and the development of malaria by E vivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygotes does not give protection against E vivaxinfection [8]

Another study among patients infected by E vivax inBrazil showed that the proportion of individuals thatdid not present the - 33TC mutation in homozygotes

Duffy bloodgroup and malaria 395

was similar among those infected by E vivax and non-Evivax If it were the case that the mutation inheterozygous form conferred any degree of protectionagainst E vivax infection one would expect toencounter a relative excess of patients without themutation among individuals infected by E vivax [75]

In vitro binding assays have proved a significantdecline in binding activity with heterozygous Duffy-positivenegative genotypes both FYAFYA null andFYBFYBnull

when compared to the homozygousDuffy-positive erythrocytes In these assays cytoad-herence between the DBP ligand domain anderythrocytes from Duffy promoter heterozygousdonors was significantly reduced [74] Previousstudies have confirmed that heterozygous Duffy-negative individuals remain susceptible to infectionsby E vivax [76] studies made by Michon et al [74]suggested that even at the heterozygous state theDuffy-negative allele can confer a quantifiableresistance advantage against E vivax

In all 23 individuals from Papua New Guinea foundwith the FYA null allele all were heterozygous(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withE vivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes from heterozygous individuals carryingthis mutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstE vivax malaria [74]

Other studies demonstrated the presence of RBCswhich react weakly with anti-Fya in eight individuals ofThai ethnicity who live in a region endemic to malaria[77] These phenotypes may occur due to the decreasein the number of RBC antigens to structuralalteration of the antigen itself or even to both motifs[77] This weak Fya antigen could in a manner similarto that observed in Africa offer a selective advantageto this population in relation to malaria as the Fyaantigen is predominant in the region

Among Indonesians who live in regions endemic tomalaria a study was carried out on the phenotypingand genotyping and the presence of the antigen Fyawith low reactivity to the anti-Fya was observed alsooccurring a discrepancy between the genotype andphenotype of these individuals suggesting anothergenetic alternative for the individuals presentingFy(a - b - ) different from the classic genetic basefor this phenotype in Mricans [37]

The presence of the Fya antigen which is weaklyreactive was also described in Malaysians [38]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null

allele Four individuals presented the Fy(a + b - )

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396 D M Langhi ampJ 0 Bardin

phenotype the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the FYB allele can confer a certain degree ofprotection against infection by Fvivax despite beingdemonstrated by Michon et al [74] a smallerexpression of the Duffy antigens in the RBCs ofindividuals with the - 33TC mutation and thatsituation could be exacerbated by the presence of themutation in both FYA and FYB allele

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

Summary

Since the molecular basis of the Duffy blood grouppolymorphisms has been determined the molecularmechanism that gives rise to the phenotypeFy(a - b - ) in black individuals has been classicallyassociated with a point mutation - 33TC in thepromoter region of a FY B allele a mutation thatwhen present in homozygosity confers protectionagainst Fvivax infection [5]

The described FYA null appears to have a morerecent origin than that of FYB null and this allelehas been reported to occur in New Guinea [7] andBrazil [8]

RBCs from individuals homozygous for the- 33TC mutation express twice the amount of Fyantigen than those heterozygous for the mutation butthe biological significance of this finding is unknown[727]

In individuals who present the GATA mutation inthe heterozygous form dose effect of Duffy antigenshas been demonstrated [25] and these data suggestthat heterozygosity for the - 33TC mutation pro-motes protection against F vivax infection butremains susceptible to it [74]

When we correlated the frequency of the - 33TCmutation and the development of malaria by Fvivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygous individuals does not give protectionagainst Fvivax infection [8]

In individuals from Papua New Guinea found tohave the FYA null allele all of them were heterozygous

(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withFvivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes in heterozygous individuals carrying thismutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstFvivax malaria [74]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null alleleFour individuals presented the Fy(a + b - ) pheno-type the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the mutation in the FYB allele can confer acertain degree of protection against infection byFvivax

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

References

[1] Mollison PL Engelfried CP Contreras M Blood transfusionin clinical medicine Oxford England Blackwell 1997

[2] Collins A Keast BJ Dracopoli N Shields DC Morton NEIntegration of gene maps Chromosome 1 Proc Nat Acad SciUSA 1992894598-4602

[3] Cutbush M Mollison PL Parkin DM A new human bloodgroup Nature 1950165188-189

[4] Sanger R Race RR Jack J The Duffy blood groups of NewYork negroes The phenotype Fy(a-b-) Br J Haematol19551370-374

[5] Tournamille C Colin Y Cartron JP Le Van Kim CDisruption of a GATA motif in the Duffy gene promoterabolishes erythroid gene expression in Duffy-negative individ-uals Nat Genet 199510224-228

[6] Chown B Lewis M Kaita H The Duffy blood group system incaucasians Evidence for a new allele Am J Hum Genet196517384-389

[7] Zimmerman PA Woolley I Masinde GL Miller SMMcNamara DT Hazlett F et al Emergence of FYA null ina Plasmodium vivax-endemic region of Papua New GuineaProc Nat Acad Sci USA 199996 13973-13977

[8] Langhi DM Albuquerque SR Covas DT Perez CA BordinJO The presence ofFYAnull allele of Duffy blood group system

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individuals whose Fy(b -) phenotype cannot beexplained by the isolated mutation in the GATA-1box [40]

Functional aspects

Although all blood group antigens are serologicallydetectable on RBCs most of them are also expressedin non-erythroid tissues raising further questions ontheir physiological function under normal andpathological conditions In addition to their structuraldiversity blood group antigens also possess widefunctional diversity and can be schematically sub-divided into five classes (a) transporters and channels(b) receptors for ligands viruses bacteria andparasites (c) adhesion molecules (d) enzymes and(e) structural proteins

Many RBCs surface molecules among those whichcarry blood groups are receptors for viruses bacteriaand parasites suggesting that these antigens may playadirect role in the pathogenesis of infectious diseases[4142]

A variety of microorganisms recognize carbohydratestructures present on glycolipids and glycoproteinsfor instance sialic acids which are abundantlyrepresented on glycophorin A (GPA) or the Cala1-4 Gal motif shared by P Pk and PI glycolipids usedby several bacterial strains and toxins responsible forupper urinary tract infections [43]

The Duffy antigens (Fyab Fy3 and Fy6) are carriedby a membrane glycoprotein exhibiting two interestingbiological properties

bull as a promiscuous receptor for chemokines of theCC (RANTES MCP-1) and CXC (IL8 mgSA)subfamilies of proinflammatory peptides namedaccording to the structure of a conserved cysteine(C) motif Hence the Duffy protein was renamedDARC for Duffy AntigenReceptor for Chemo-kines

bull as erythroid receptor for R vivax and R knowlesi

Duffy antigen receptor for chemokine

The fact that RBCs possess a chemokine receptor wasfirst established by Darbone et al [44]

Chemokines constitute a family of proinflammatorycytokines capable of activating leukocytes and causingchemotaxis but other important functions have beendiscovered including angiogenic and angiostaticactivities [45] The number and spacing of amino-terminal cysteines have been used in the classificationof chemokines into four families C CC CXC andCXXXC The biological effect of chemokines ismediated by the binding and activation of G-protein-coupled seven-transmembrane domain chemokinereceptors [46] Some chemokines (classes CC and

Duffy blood group and malaria 393

CXC) also bind to the Duffy blood group glyco-protein which is now called DARC

Of the cellular chemokine receptors CXCR4CCR5 and Duffy are the only receptors that havebeen unequivocally proven to act as coreceptors forcell entry of pathogens Duffy for the entry of malarialparasite R vivax and CCR5 for the entry of M-tropicstrains of human immunodeficiency virus (HIV) [47]

During experiments with chemokine receptors inerythrocytes the perfect correlation between thebonding of the chemokines to the erythrocytes andthe presence of the Duffy antigen was well established[12] It has been demonstrated that anti-Fy antibodiesinhibited the chemokine binding ability to Duffypositive erythrocytes and chemokines that bind toerythrocytes block RBC invasion by malarial parasitesthat use the Duffy antigen as a receptor [12]

CC and CXC chemokines bind to DARC with highaffinity suggesting some role in inflammatory reactions[31] DARC might play a role as a scavenger on theRBC surface to eliminate excess of toxic chemokinesproduced in some pathologic situations [44]

There are some experimental studies suggestingthat DARC is a redundant protein that may playa rolein the regulation of induced leukocyte trafficking invivo [48]

The CXCR4 and CCR5 chemokine receptors aremandatory cofactors for HIV infection by interactingwith the viral envelope gp 120 in the presence of CD4[49] A report shows that HIV-1 viral particles mightbind to the Fy protein suggesting that RBCs mayfunction as a virus reservoir or as a receptor for theentry in some cells [50]

Duffy antigen receptor for R vivax

R vivax causes approximately between 70 and 80million cases of malaria per year and is the most amplydistributed human malaria in the world [51]

One of the most interesting aspects of the Duffyantigen is its function as a receptor for the humanmalaria parasite R vivax

In 1975 Miller et al [9] showed that Duffy-negative human RBCs were resistant to invasion byR knowlesi a monkey malaria parasite that was knownto be capable of invading human RBCs and in rareinstances of infecting humans Furthermore anti-Fyaand anti-Fyb blocked invasion of R knowlesi intoFy(a + b -) and Fy(a - b +) RBCs respectivelyand the treatment of the RBCs with enzymes whichremove from their surface the antigenic determinantsFya e Fyb (chemotrypsin and pronase) making theRBCs resistant to invasion [9]

Studies on R knowlesi were extended to R vivax ahuman malaria that is second only to Plasmodiumfaleiparum in terms of the toll it takes on populationsof endemic areas Although R vivax is widespreadthroughout the tropical and subtropical world and

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is a major drain on the health care resources of Indiaand Southeast Asia it is absent from West Africawhere more than 95 of the population is Duffynegative Miller et al [52] showed that resistance toR vivax correlates with the Duffy-negative pheno-type and more recently Barnwell et al [53] showedthat R vivax merozoites are incapable of invadingDuffy-negative RBCs Thus R vivax like R knowlesirelies on a Duffy antigen-parasite ligand interactionsfor invasion It should be noted that R vivaxpreferentially invades reticulocytes [54] althoughmature RBCs as well as reticulocytes express acoreceptor that along with the Duffy antigen isnecessary for optimal invasion by R vivax Thebonding agent of the R vivax parasite whichspecifically bonds itself to the reticulocytes was alsoidentified and the cDNA encoding this protein wascloned [54]

When the anti-Fy6 antibody became availablestudies demonstrated that the presence of Fy6 is whatresults from the invasion of human RBCs by themerozoites of the R vivax [53]

In humans the presence or absence of Fy6correlates with the presence or absence of the Fyaand Fyb In other words the Fy(a +) or Fy(b + )RBCs are always Fy6 positive and Fy(a - b - ) RBCsare Fy6 negative The invasion of the Fy(a + ) RBCsby R vivax can be partially blocked by the covering ofthe RBC with anti-Fya [9] and totally blocked bycovering the RBC with anti-Fy6 [55]

The fact that the great majority of black African andAfro-American individuals are resistant to infection byR vivax is directly related to the Fy(a - b - )phenotype or in other words the absence of the Fyaand Fyb antigens

One Duffy binding protein of R vivax calledPvDAP-l (Pv - indicates R vivax and DAPindicates Duffy-associating protein) has a criticalrole as a bonder in the adhesion of the parasite andposterior invasion of the positive Duffy RBCs [56] Rvivax as with other species of human Plasmodiuminitiate erythrocyte invasion through the expression ofvarious surface organelles and structures on themerozoite which bind with the surface proteins ofthe erythrocyte The well-characterized interactionof the bonder-receptor involves the Duffy bondingprotein expressed in the merozoite form of theR vivax and its corresponding receptor in theerythrocyte the receptor DARC This interaction isunique among human malaria infections where theinteraction of the bonding receptor is essential to theinvasion of the erythrocyte by R vivax Alternatemodels of erythrocytic invasion have not yet beendescribed [57] This Duffy bonding protein ofR vivax belongs to the family of bonding proteinsfor erythrocytes which also includes the bondingprotein for sialic acid R jalciparum and to the Duffybonding protein R knowlesi

R vivax merozoites invade reticulocytes that expressthe Duffy protein and the counter receptor is a140 kDa protein (PvDBP) that belongs to Plasmodiumadhesion proteins including EBA-175 and PfEMPl(see above) all having in the NHz-ter region acysteine-rich domain (called DPP region II) mediat-ing erythrocyte binding [5859]

It is not known whether the Duffy antigen is astructural junction component because the molecularnature of junction has yet to be determined It is clearhowever that the antigen interaction of the parasiteDuffy-receptor is crucial to the formation of thejunction and subsequent invasion [57]

Several blood group molecules of red cell surfacecontribute to the complex mechanisms of invasion andsequestration steps that characterize the most severeform of malaria caused by R jalciparum R jalciparummerozoites may invade RBCs through sialic acid-dependent and independent pathways [6061] GPA isinvolved in the sialic-acid dependent pathway and thecounter receptor of GPA is the parasite proteinerythrocyte binding antigen (EBA) of 175 kDa [62]which binds to sialic acid and peptide backbone ofGPAthrough a cysteine-rich domain (region II) analogousto the one mediating R vivaxDuffy protein interaction[63] Recently a homologue to EBA-175 calledBAEBL has been shown to use GPC for invasion[64] Rosette formation and cytoadherence ofR jalciparum-infected erythrocytes to vascular endo-thelium result in the sequestration of infected cellsparticularly in the brain vasculature (resulting inanoxia altered brain function and coma) and thusrepresent a major cause of cerebral malaria [65]However the role of the immune system is likely also tobe of critical importance [66] Cytoadherence ofRBCscontaining mature-stage parasites to vascular endo-thelium and platelets is a protective mechanism used bythe parasite to avoid elimination This is mediated byseveral membrane proteins receptors present onendothelial cells (CD36 ICAM-l PECAM-l TSPchondroitin sulfate) and cryptic antigens ofRBCs likethose exposed on altered Band 3 [67] The parasitizedRBCs not only adhere to vascular endothelium but alsoadhere to uninfected RBCs a process known asrosette formation This is mainly mediated by thecomplement receptor CRI (CD35 carrier of Knoppsantigens) since among a large series of null varianterythrocytes tested for the ability to form rosettes onlythose of the Helgenson phenotype (CRI-deficient)were negative In addition Sla( - ) red cells which havea reduced copy number of CRl rosetted poorly [68]The counter receptor of CRI is PfEMP 1 (R jalciparumErythrocyte Membrane Protein 1) a protein of 300-350 kDa produced by a gene of the varfamily but otherligands of PfEMP 1 (heparin sulfate glycans ABOgroups CD36) are also involved in rosette formation[69] The main function of CRI (190-280 kDa) is tocapture and remove from the liver and spleen immune

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complexes containing C3b and C4b but it also plays arole in the immune response [70] The CR1 level onRBCs is low but may vary widely The extramembranous region of CR1 is made of 30 shortconsensus repeats (SCR) and two Knopps blood grouppolymorphisms have been assigned to the homologousregion D within SCR24 (McCab = K1590E) andSCR25 (SlaNil = R160 1G) [71]

Duffy polymorphism and malaria

Malaria is an important selective force for humangenetic adaptations due to the sustained lethal impactthat it has had on human populations around theworld Homozygosity for the Duffy-negative bloodgroup antigen confers complete resistance to vivaxmalaria Nevertheless it is unclear whether selectivepressure of vivax malaria alone was the main cause forthe emergence and fixation of the FYB null FYB null

genotype in much of West Africa where vivax malariais absent Indeed there is little doubt that malariacaused by E vivax is not as directly lethal asE jalciparum but a fulminant E vivax infection stillcauses serious morbidity in those living at theminimum subsistence level The emergence of thenew FYA allele carrying the - 33TC mutation inE vivax-endemic region of Papua New Guineafurther supported the hypothesis that E vivax malariacan act as a selective agent of the Fy(a - b - )phenotypes in this region [7]

Accordingly Fy mRNA and DARC are normallyexpressed in nonerythroid tissues in populations ofWest Mrica where E vivax is no longer present It isassumed that the most common phenotypeFy(a - b - ) represents an adaptive response to resistmalarial infection [325]

RBCs from individuals homozygous for the wild-type promoter (Genotypes FYAFYA FYBFYB andFYAFYB) express twice the amount of Fy antigenthan those heterozygous for the GATA-1 mutation(genotypes FYAFYB SE FYBFYB SE) [727] but thebiological significance of this finding is unknown [7]

In individuals who present the GATA mutation inthe heterozygous form dose effect has been demon-strated being that only 50 of the Duffy antigens areexpressed in the erythrocytes [73] These data suggestthat heterozygosity for the - 33TC mutation in theallele promotes the protection against E vivaxinfection but is still susceptible to it [74]

When we correlated the frequency of - 33TCmutation and the development of malaria by E vivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygotes does not give protection against E vivaxinfection [8]

Another study among patients infected by E vivax inBrazil showed that the proportion of individuals thatdid not present the - 33TC mutation in homozygotes

Duffy bloodgroup and malaria 395

was similar among those infected by E vivax and non-Evivax If it were the case that the mutation inheterozygous form conferred any degree of protectionagainst E vivax infection one would expect toencounter a relative excess of patients without themutation among individuals infected by E vivax [75]

In vitro binding assays have proved a significantdecline in binding activity with heterozygous Duffy-positivenegative genotypes both FYAFYA null andFYBFYBnull

when compared to the homozygousDuffy-positive erythrocytes In these assays cytoad-herence between the DBP ligand domain anderythrocytes from Duffy promoter heterozygousdonors was significantly reduced [74] Previousstudies have confirmed that heterozygous Duffy-negative individuals remain susceptible to infectionsby E vivax [76] studies made by Michon et al [74]suggested that even at the heterozygous state theDuffy-negative allele can confer a quantifiableresistance advantage against E vivax

In all 23 individuals from Papua New Guinea foundwith the FYA null allele all were heterozygous(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withE vivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes from heterozygous individuals carryingthis mutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstE vivax malaria [74]

Other studies demonstrated the presence of RBCswhich react weakly with anti-Fya in eight individuals ofThai ethnicity who live in a region endemic to malaria[77] These phenotypes may occur due to the decreasein the number of RBC antigens to structuralalteration of the antigen itself or even to both motifs[77] This weak Fya antigen could in a manner similarto that observed in Africa offer a selective advantageto this population in relation to malaria as the Fyaantigen is predominant in the region

Among Indonesians who live in regions endemic tomalaria a study was carried out on the phenotypingand genotyping and the presence of the antigen Fyawith low reactivity to the anti-Fya was observed alsooccurring a discrepancy between the genotype andphenotype of these individuals suggesting anothergenetic alternative for the individuals presentingFy(a - b - ) different from the classic genetic basefor this phenotype in Mricans [37]

The presence of the Fya antigen which is weaklyreactive was also described in Malaysians [38]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null

allele Four individuals presented the Fy(a + b - )

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phenotype the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the FYB allele can confer a certain degree ofprotection against infection by Fvivax despite beingdemonstrated by Michon et al [74] a smallerexpression of the Duffy antigens in the RBCs ofindividuals with the - 33TC mutation and thatsituation could be exacerbated by the presence of themutation in both FYA and FYB allele

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

Summary

Since the molecular basis of the Duffy blood grouppolymorphisms has been determined the molecularmechanism that gives rise to the phenotypeFy(a - b - ) in black individuals has been classicallyassociated with a point mutation - 33TC in thepromoter region of a FY B allele a mutation thatwhen present in homozygosity confers protectionagainst Fvivax infection [5]

The described FYA null appears to have a morerecent origin than that of FYB null and this allelehas been reported to occur in New Guinea [7] andBrazil [8]

RBCs from individuals homozygous for the- 33TC mutation express twice the amount of Fyantigen than those heterozygous for the mutation butthe biological significance of this finding is unknown[727]

In individuals who present the GATA mutation inthe heterozygous form dose effect of Duffy antigenshas been demonstrated [25] and these data suggestthat heterozygosity for the - 33TC mutation pro-motes protection against F vivax infection butremains susceptible to it [74]

When we correlated the frequency of the - 33TCmutation and the development of malaria by Fvivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygous individuals does not give protectionagainst Fvivax infection [8]

In individuals from Papua New Guinea found tohave the FYA null allele all of them were heterozygous

(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withFvivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes in heterozygous individuals carrying thismutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstFvivax malaria [74]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null alleleFour individuals presented the Fy(a + b - ) pheno-type the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the mutation in the FYB allele can confer acertain degree of protection against infection byFvivax

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

References

[1] Mollison PL Engelfried CP Contreras M Blood transfusionin clinical medicine Oxford England Blackwell 1997

[2] Collins A Keast BJ Dracopoli N Shields DC Morton NEIntegration of gene maps Chromosome 1 Proc Nat Acad SciUSA 1992894598-4602

[3] Cutbush M Mollison PL Parkin DM A new human bloodgroup Nature 1950165188-189

[4] Sanger R Race RR Jack J The Duffy blood groups of NewYork negroes The phenotype Fy(a-b-) Br J Haematol19551370-374

[5] Tournamille C Colin Y Cartron JP Le Van Kim CDisruption of a GATA motif in the Duffy gene promoterabolishes erythroid gene expression in Duffy-negative individ-uals Nat Genet 199510224-228

[6] Chown B Lewis M Kaita H The Duffy blood group system incaucasians Evidence for a new allele Am J Hum Genet196517384-389

[7] Zimmerman PA Woolley I Masinde GL Miller SMMcNamara DT Hazlett F et al Emergence of FYA null ina Plasmodium vivax-endemic region of Papua New GuineaProc Nat Acad Sci USA 199996 13973-13977

[8] Langhi DM Albuquerque SR Covas DT Perez CA BordinJO The presence ofFYAnull allele of Duffy blood group system

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is a major drain on the health care resources of Indiaand Southeast Asia it is absent from West Africawhere more than 95 of the population is Duffynegative Miller et al [52] showed that resistance toR vivax correlates with the Duffy-negative pheno-type and more recently Barnwell et al [53] showedthat R vivax merozoites are incapable of invadingDuffy-negative RBCs Thus R vivax like R knowlesirelies on a Duffy antigen-parasite ligand interactionsfor invasion It should be noted that R vivaxpreferentially invades reticulocytes [54] althoughmature RBCs as well as reticulocytes express acoreceptor that along with the Duffy antigen isnecessary for optimal invasion by R vivax Thebonding agent of the R vivax parasite whichspecifically bonds itself to the reticulocytes was alsoidentified and the cDNA encoding this protein wascloned [54]

When the anti-Fy6 antibody became availablestudies demonstrated that the presence of Fy6 is whatresults from the invasion of human RBCs by themerozoites of the R vivax [53]

In humans the presence or absence of Fy6correlates with the presence or absence of the Fyaand Fyb In other words the Fy(a +) or Fy(b + )RBCs are always Fy6 positive and Fy(a - b - ) RBCsare Fy6 negative The invasion of the Fy(a + ) RBCsby R vivax can be partially blocked by the covering ofthe RBC with anti-Fya [9] and totally blocked bycovering the RBC with anti-Fy6 [55]

The fact that the great majority of black African andAfro-American individuals are resistant to infection byR vivax is directly related to the Fy(a - b - )phenotype or in other words the absence of the Fyaand Fyb antigens

One Duffy binding protein of R vivax calledPvDAP-l (Pv - indicates R vivax and DAPindicates Duffy-associating protein) has a criticalrole as a bonder in the adhesion of the parasite andposterior invasion of the positive Duffy RBCs [56] Rvivax as with other species of human Plasmodiuminitiate erythrocyte invasion through the expression ofvarious surface organelles and structures on themerozoite which bind with the surface proteins ofthe erythrocyte The well-characterized interactionof the bonder-receptor involves the Duffy bondingprotein expressed in the merozoite form of theR vivax and its corresponding receptor in theerythrocyte the receptor DARC This interaction isunique among human malaria infections where theinteraction of the bonding receptor is essential to theinvasion of the erythrocyte by R vivax Alternatemodels of erythrocytic invasion have not yet beendescribed [57] This Duffy bonding protein ofR vivax belongs to the family of bonding proteinsfor erythrocytes which also includes the bondingprotein for sialic acid R jalciparum and to the Duffybonding protein R knowlesi

R vivax merozoites invade reticulocytes that expressthe Duffy protein and the counter receptor is a140 kDa protein (PvDBP) that belongs to Plasmodiumadhesion proteins including EBA-175 and PfEMPl(see above) all having in the NHz-ter region acysteine-rich domain (called DPP region II) mediat-ing erythrocyte binding [5859]

It is not known whether the Duffy antigen is astructural junction component because the molecularnature of junction has yet to be determined It is clearhowever that the antigen interaction of the parasiteDuffy-receptor is crucial to the formation of thejunction and subsequent invasion [57]

Several blood group molecules of red cell surfacecontribute to the complex mechanisms of invasion andsequestration steps that characterize the most severeform of malaria caused by R jalciparum R jalciparummerozoites may invade RBCs through sialic acid-dependent and independent pathways [6061] GPA isinvolved in the sialic-acid dependent pathway and thecounter receptor of GPA is the parasite proteinerythrocyte binding antigen (EBA) of 175 kDa [62]which binds to sialic acid and peptide backbone ofGPAthrough a cysteine-rich domain (region II) analogousto the one mediating R vivaxDuffy protein interaction[63] Recently a homologue to EBA-175 calledBAEBL has been shown to use GPC for invasion[64] Rosette formation and cytoadherence ofR jalciparum-infected erythrocytes to vascular endo-thelium result in the sequestration of infected cellsparticularly in the brain vasculature (resulting inanoxia altered brain function and coma) and thusrepresent a major cause of cerebral malaria [65]However the role of the immune system is likely also tobe of critical importance [66] Cytoadherence ofRBCscontaining mature-stage parasites to vascular endo-thelium and platelets is a protective mechanism used bythe parasite to avoid elimination This is mediated byseveral membrane proteins receptors present onendothelial cells (CD36 ICAM-l PECAM-l TSPchondroitin sulfate) and cryptic antigens ofRBCs likethose exposed on altered Band 3 [67] The parasitizedRBCs not only adhere to vascular endothelium but alsoadhere to uninfected RBCs a process known asrosette formation This is mainly mediated by thecomplement receptor CRI (CD35 carrier of Knoppsantigens) since among a large series of null varianterythrocytes tested for the ability to form rosettes onlythose of the Helgenson phenotype (CRI-deficient)were negative In addition Sla( - ) red cells which havea reduced copy number of CRl rosetted poorly [68]The counter receptor of CRI is PfEMP 1 (R jalciparumErythrocyte Membrane Protein 1) a protein of 300-350 kDa produced by a gene of the varfamily but otherligands of PfEMP 1 (heparin sulfate glycans ABOgroups CD36) are also involved in rosette formation[69] The main function of CRI (190-280 kDa) is tocapture and remove from the liver and spleen immune

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complexes containing C3b and C4b but it also plays arole in the immune response [70] The CR1 level onRBCs is low but may vary widely The extramembranous region of CR1 is made of 30 shortconsensus repeats (SCR) and two Knopps blood grouppolymorphisms have been assigned to the homologousregion D within SCR24 (McCab = K1590E) andSCR25 (SlaNil = R160 1G) [71]

Duffy polymorphism and malaria

Malaria is an important selective force for humangenetic adaptations due to the sustained lethal impactthat it has had on human populations around theworld Homozygosity for the Duffy-negative bloodgroup antigen confers complete resistance to vivaxmalaria Nevertheless it is unclear whether selectivepressure of vivax malaria alone was the main cause forthe emergence and fixation of the FYB null FYB null

genotype in much of West Africa where vivax malariais absent Indeed there is little doubt that malariacaused by E vivax is not as directly lethal asE jalciparum but a fulminant E vivax infection stillcauses serious morbidity in those living at theminimum subsistence level The emergence of thenew FYA allele carrying the - 33TC mutation inE vivax-endemic region of Papua New Guineafurther supported the hypothesis that E vivax malariacan act as a selective agent of the Fy(a - b - )phenotypes in this region [7]

Accordingly Fy mRNA and DARC are normallyexpressed in nonerythroid tissues in populations ofWest Mrica where E vivax is no longer present It isassumed that the most common phenotypeFy(a - b - ) represents an adaptive response to resistmalarial infection [325]

RBCs from individuals homozygous for the wild-type promoter (Genotypes FYAFYA FYBFYB andFYAFYB) express twice the amount of Fy antigenthan those heterozygous for the GATA-1 mutation(genotypes FYAFYB SE FYBFYB SE) [727] but thebiological significance of this finding is unknown [7]

In individuals who present the GATA mutation inthe heterozygous form dose effect has been demon-strated being that only 50 of the Duffy antigens areexpressed in the erythrocytes [73] These data suggestthat heterozygosity for the - 33TC mutation in theallele promotes the protection against E vivaxinfection but is still susceptible to it [74]

When we correlated the frequency of - 33TCmutation and the development of malaria by E vivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygotes does not give protection against E vivaxinfection [8]

Another study among patients infected by E vivax inBrazil showed that the proportion of individuals thatdid not present the - 33TC mutation in homozygotes

Duffy bloodgroup and malaria 395

was similar among those infected by E vivax and non-Evivax If it were the case that the mutation inheterozygous form conferred any degree of protectionagainst E vivax infection one would expect toencounter a relative excess of patients without themutation among individuals infected by E vivax [75]

In vitro binding assays have proved a significantdecline in binding activity with heterozygous Duffy-positivenegative genotypes both FYAFYA null andFYBFYBnull

when compared to the homozygousDuffy-positive erythrocytes In these assays cytoad-herence between the DBP ligand domain anderythrocytes from Duffy promoter heterozygousdonors was significantly reduced [74] Previousstudies have confirmed that heterozygous Duffy-negative individuals remain susceptible to infectionsby E vivax [76] studies made by Michon et al [74]suggested that even at the heterozygous state theDuffy-negative allele can confer a quantifiableresistance advantage against E vivax

In all 23 individuals from Papua New Guinea foundwith the FYA null allele all were heterozygous(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withE vivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes from heterozygous individuals carryingthis mutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstE vivax malaria [74]

Other studies demonstrated the presence of RBCswhich react weakly with anti-Fya in eight individuals ofThai ethnicity who live in a region endemic to malaria[77] These phenotypes may occur due to the decreasein the number of RBC antigens to structuralalteration of the antigen itself or even to both motifs[77] This weak Fya antigen could in a manner similarto that observed in Africa offer a selective advantageto this population in relation to malaria as the Fyaantigen is predominant in the region

Among Indonesians who live in regions endemic tomalaria a study was carried out on the phenotypingand genotyping and the presence of the antigen Fyawith low reactivity to the anti-Fya was observed alsooccurring a discrepancy between the genotype andphenotype of these individuals suggesting anothergenetic alternative for the individuals presentingFy(a - b - ) different from the classic genetic basefor this phenotype in Mricans [37]

The presence of the Fya antigen which is weaklyreactive was also described in Malaysians [38]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null

allele Four individuals presented the Fy(a + b - )

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phenotype the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the FYB allele can confer a certain degree ofprotection against infection by Fvivax despite beingdemonstrated by Michon et al [74] a smallerexpression of the Duffy antigens in the RBCs ofindividuals with the - 33TC mutation and thatsituation could be exacerbated by the presence of themutation in both FYA and FYB allele

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

Summary

Since the molecular basis of the Duffy blood grouppolymorphisms has been determined the molecularmechanism that gives rise to the phenotypeFy(a - b - ) in black individuals has been classicallyassociated with a point mutation - 33TC in thepromoter region of a FY B allele a mutation thatwhen present in homozygosity confers protectionagainst Fvivax infection [5]

The described FYA null appears to have a morerecent origin than that of FYB null and this allelehas been reported to occur in New Guinea [7] andBrazil [8]

RBCs from individuals homozygous for the- 33TC mutation express twice the amount of Fyantigen than those heterozygous for the mutation butthe biological significance of this finding is unknown[727]

In individuals who present the GATA mutation inthe heterozygous form dose effect of Duffy antigenshas been demonstrated [25] and these data suggestthat heterozygosity for the - 33TC mutation pro-motes protection against F vivax infection butremains susceptible to it [74]

When we correlated the frequency of the - 33TCmutation and the development of malaria by Fvivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygous individuals does not give protectionagainst Fvivax infection [8]

In individuals from Papua New Guinea found tohave the FYA null allele all of them were heterozygous

(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withFvivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes in heterozygous individuals carrying thismutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstFvivax malaria [74]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null alleleFour individuals presented the Fy(a + b - ) pheno-type the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the mutation in the FYB allele can confer acertain degree of protection against infection byFvivax

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

References

[1] Mollison PL Engelfried CP Contreras M Blood transfusionin clinical medicine Oxford England Blackwell 1997

[2] Collins A Keast BJ Dracopoli N Shields DC Morton NEIntegration of gene maps Chromosome 1 Proc Nat Acad SciUSA 1992894598-4602

[3] Cutbush M Mollison PL Parkin DM A new human bloodgroup Nature 1950165188-189

[4] Sanger R Race RR Jack J The Duffy blood groups of NewYork negroes The phenotype Fy(a-b-) Br J Haematol19551370-374

[5] Tournamille C Colin Y Cartron JP Le Van Kim CDisruption of a GATA motif in the Duffy gene promoterabolishes erythroid gene expression in Duffy-negative individ-uals Nat Genet 199510224-228

[6] Chown B Lewis M Kaita H The Duffy blood group system incaucasians Evidence for a new allele Am J Hum Genet196517384-389

[7] Zimmerman PA Woolley I Masinde GL Miller SMMcNamara DT Hazlett F et al Emergence of FYA null ina Plasmodium vivax-endemic region of Papua New GuineaProc Nat Acad Sci USA 199996 13973-13977

[8] Langhi DM Albuquerque SR Covas DT Perez CA BordinJO The presence ofFYAnull allele of Duffy blood group system

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complexes containing C3b and C4b but it also plays arole in the immune response [70] The CR1 level onRBCs is low but may vary widely The extramembranous region of CR1 is made of 30 shortconsensus repeats (SCR) and two Knopps blood grouppolymorphisms have been assigned to the homologousregion D within SCR24 (McCab = K1590E) andSCR25 (SlaNil = R160 1G) [71]

Duffy polymorphism and malaria

Malaria is an important selective force for humangenetic adaptations due to the sustained lethal impactthat it has had on human populations around theworld Homozygosity for the Duffy-negative bloodgroup antigen confers complete resistance to vivaxmalaria Nevertheless it is unclear whether selectivepressure of vivax malaria alone was the main cause forthe emergence and fixation of the FYB null FYB null

genotype in much of West Africa where vivax malariais absent Indeed there is little doubt that malariacaused by E vivax is not as directly lethal asE jalciparum but a fulminant E vivax infection stillcauses serious morbidity in those living at theminimum subsistence level The emergence of thenew FYA allele carrying the - 33TC mutation inE vivax-endemic region of Papua New Guineafurther supported the hypothesis that E vivax malariacan act as a selective agent of the Fy(a - b - )phenotypes in this region [7]

Accordingly Fy mRNA and DARC are normallyexpressed in nonerythroid tissues in populations ofWest Mrica where E vivax is no longer present It isassumed that the most common phenotypeFy(a - b - ) represents an adaptive response to resistmalarial infection [325]

RBCs from individuals homozygous for the wild-type promoter (Genotypes FYAFYA FYBFYB andFYAFYB) express twice the amount of Fy antigenthan those heterozygous for the GATA-1 mutation(genotypes FYAFYB SE FYBFYB SE) [727] but thebiological significance of this finding is unknown [7]

In individuals who present the GATA mutation inthe heterozygous form dose effect has been demon-strated being that only 50 of the Duffy antigens areexpressed in the erythrocytes [73] These data suggestthat heterozygosity for the - 33TC mutation in theallele promotes the protection against E vivaxinfection but is still susceptible to it [74]

When we correlated the frequency of - 33TCmutation and the development of malaria by E vivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygotes does not give protection against E vivaxinfection [8]

Another study among patients infected by E vivax inBrazil showed that the proportion of individuals thatdid not present the - 33TC mutation in homozygotes

Duffy bloodgroup and malaria 395

was similar among those infected by E vivax and non-Evivax If it were the case that the mutation inheterozygous form conferred any degree of protectionagainst E vivax infection one would expect toencounter a relative excess of patients without themutation among individuals infected by E vivax [75]

In vitro binding assays have proved a significantdecline in binding activity with heterozygous Duffy-positivenegative genotypes both FYAFYA null andFYBFYBnull

when compared to the homozygousDuffy-positive erythrocytes In these assays cytoad-herence between the DBP ligand domain anderythrocytes from Duffy promoter heterozygousdonors was significantly reduced [74] Previousstudies have confirmed that heterozygous Duffy-negative individuals remain susceptible to infectionsby E vivax [76] studies made by Michon et al [74]suggested that even at the heterozygous state theDuffy-negative allele can confer a quantifiableresistance advantage against E vivax

In all 23 individuals from Papua New Guinea foundwith the FYA null allele all were heterozygous(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withE vivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes from heterozygous individuals carryingthis mutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstE vivax malaria [74]

Other studies demonstrated the presence of RBCswhich react weakly with anti-Fya in eight individuals ofThai ethnicity who live in a region endemic to malaria[77] These phenotypes may occur due to the decreasein the number of RBC antigens to structuralalteration of the antigen itself or even to both motifs[77] This weak Fya antigen could in a manner similarto that observed in Africa offer a selective advantageto this population in relation to malaria as the Fyaantigen is predominant in the region

Among Indonesians who live in regions endemic tomalaria a study was carried out on the phenotypingand genotyping and the presence of the antigen Fyawith low reactivity to the anti-Fya was observed alsooccurring a discrepancy between the genotype andphenotype of these individuals suggesting anothergenetic alternative for the individuals presentingFy(a - b - ) different from the classic genetic basefor this phenotype in Mricans [37]

The presence of the Fya antigen which is weaklyreactive was also described in Malaysians [38]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null

allele Four individuals presented the Fy(a + b - )

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phenotype the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the FYB allele can confer a certain degree ofprotection against infection by Fvivax despite beingdemonstrated by Michon et al [74] a smallerexpression of the Duffy antigens in the RBCs ofindividuals with the - 33TC mutation and thatsituation could be exacerbated by the presence of themutation in both FYA and FYB allele

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

Summary

Since the molecular basis of the Duffy blood grouppolymorphisms has been determined the molecularmechanism that gives rise to the phenotypeFy(a - b - ) in black individuals has been classicallyassociated with a point mutation - 33TC in thepromoter region of a FY B allele a mutation thatwhen present in homozygosity confers protectionagainst Fvivax infection [5]

The described FYA null appears to have a morerecent origin than that of FYB null and this allelehas been reported to occur in New Guinea [7] andBrazil [8]

RBCs from individuals homozygous for the- 33TC mutation express twice the amount of Fyantigen than those heterozygous for the mutation butthe biological significance of this finding is unknown[727]

In individuals who present the GATA mutation inthe heterozygous form dose effect of Duffy antigenshas been demonstrated [25] and these data suggestthat heterozygosity for the - 33TC mutation pro-motes protection against F vivax infection butremains susceptible to it [74]

When we correlated the frequency of the - 33TCmutation and the development of malaria by Fvivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygous individuals does not give protectionagainst Fvivax infection [8]

In individuals from Papua New Guinea found tohave the FYA null allele all of them were heterozygous

(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withFvivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes in heterozygous individuals carrying thismutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstFvivax malaria [74]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null alleleFour individuals presented the Fy(a + b - ) pheno-type the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the mutation in the FYB allele can confer acertain degree of protection against infection byFvivax

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

References

[1] Mollison PL Engelfried CP Contreras M Blood transfusionin clinical medicine Oxford England Blackwell 1997

[2] Collins A Keast BJ Dracopoli N Shields DC Morton NEIntegration of gene maps Chromosome 1 Proc Nat Acad SciUSA 1992894598-4602

[3] Cutbush M Mollison PL Parkin DM A new human bloodgroup Nature 1950165188-189

[4] Sanger R Race RR Jack J The Duffy blood groups of NewYork negroes The phenotype Fy(a-b-) Br J Haematol19551370-374

[5] Tournamille C Colin Y Cartron JP Le Van Kim CDisruption of a GATA motif in the Duffy gene promoterabolishes erythroid gene expression in Duffy-negative individ-uals Nat Genet 199510224-228

[6] Chown B Lewis M Kaita H The Duffy blood group system incaucasians Evidence for a new allele Am J Hum Genet196517384-389

[7] Zimmerman PA Woolley I Masinde GL Miller SMMcNamara DT Hazlett F et al Emergence of FYA null ina Plasmodium vivax-endemic region of Papua New GuineaProc Nat Acad Sci USA 199996 13973-13977

[8] Langhi DM Albuquerque SR Covas DT Perez CA BordinJO The presence ofFYAnull allele of Duffy blood group system

Pub

lishe

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ey P

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396 D M Langhi ampJ 0 Bardin

phenotype the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the FYB allele can confer a certain degree ofprotection against infection by Fvivax despite beingdemonstrated by Michon et al [74] a smallerexpression of the Duffy antigens in the RBCs ofindividuals with the - 33TC mutation and thatsituation could be exacerbated by the presence of themutation in both FYA and FYB allele

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

Summary

Since the molecular basis of the Duffy blood grouppolymorphisms has been determined the molecularmechanism that gives rise to the phenotypeFy(a - b - ) in black individuals has been classicallyassociated with a point mutation - 33TC in thepromoter region of a FY B allele a mutation thatwhen present in homozygosity confers protectionagainst Fvivax infection [5]

The described FYA null appears to have a morerecent origin than that of FYB null and this allelehas been reported to occur in New Guinea [7] andBrazil [8]

RBCs from individuals homozygous for the- 33TC mutation express twice the amount of Fyantigen than those heterozygous for the mutation butthe biological significance of this finding is unknown[727]

In individuals who present the GATA mutation inthe heterozygous form dose effect of Duffy antigenshas been demonstrated [25] and these data suggestthat heterozygosity for the - 33TC mutation pro-motes protection against F vivax infection butremains susceptible to it [74]

When we correlated the frequency of the - 33TCmutation and the development of malaria by Fvivaxin individuals from a malarial endemic region inBrazil the data suggest that the - 33TC mutation inheterozygous individuals does not give protectionagainst Fvivax infection [8]

In individuals from Papua New Guinea found tohave the FYA null allele all of them were heterozygous

(FYAFYA null) for the allele Flow cytometric analysisrevealed that these individuals expressed half theamount of Duffy antigen on their erythrocytesindicating a gene dosage effect A definitive conclusioncould not be made on whether these heterozygousindividuals were less susceptible to infection withFvivax [74] However it has been demonstrated thatPvDBP adherence to RBCs is significantly reduced forerythrocytes in heterozygous individuals carrying thismutation suggesting that this new allelic form ofDuffy negativity is correlated with resistance againstFvivax malaria [74]

In our study 5 individuals from malaria endemicregion in the Amazon presented the FYA null alleleFour individuals presented the Fy(a + b - ) pheno-type the FYAFYB genotype and the - 33TCmutation in the homozygous form and one individualpresented the Fy(a + b - ) phenotype the FYAFYAgenotype and the - 33TC mutation in the hetero-zygous form Of the four individuals with theFy(a + b -) phenotype and FYAFYB genotypetwo never acquired malaria one acquired malaria byFfaleiparum and one did not inform his epidemiolo-gical status for malaria The individual who presentedthe Fy(a + b - ) phenotype and FYAFYA genotypehad acquired malaria by Fvivax and Ffaleiparum [8]Therefore due to the small number of cases wecannot affirm that the presence of the - 33TCmutation in the FYA allele isolated or in associationwith the mutation in the FYB allele can confer acertain degree of protection against infection byFvivax

No data on F vivax susceptibility are currentlyavailable for individuals that express the FYB weak

allele

References

[1] Mollison PL Engelfried CP Contreras M Blood transfusionin clinical medicine Oxford England Blackwell 1997

[2] Collins A Keast BJ Dracopoli N Shields DC Morton NEIntegration of gene maps Chromosome 1 Proc Nat Acad SciUSA 1992894598-4602

[3] Cutbush M Mollison PL Parkin DM A new human bloodgroup Nature 1950165188-189

[4] Sanger R Race RR Jack J The Duffy blood groups of NewYork negroes The phenotype Fy(a-b-) Br J Haematol19551370-374

[5] Tournamille C Colin Y Cartron JP Le Van Kim CDisruption of a GATA motif in the Duffy gene promoterabolishes erythroid gene expression in Duffy-negative individ-uals Nat Genet 199510224-228

[6] Chown B Lewis M Kaita H The Duffy blood group system incaucasians Evidence for a new allele Am J Hum Genet196517384-389

[7] Zimmerman PA Woolley I Masinde GL Miller SMMcNamara DT Hazlett F et al Emergence of FYA null ina Plasmodium vivax-endemic region of Papua New GuineaProc Nat Acad Sci USA 199996 13973-13977

[8] Langhi DM Albuquerque SR Covas DT Perez CA BordinJO The presence ofFYAnull allele of Duffy blood group system

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