Hematology, October/December

2006; 11(5/6): 389-398

informa
healthcare

Duffy blood group and malaria

DANTE M. LANGHI Jr! & JOSE ORLANDO BORDIN2

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

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

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Abstract Very important progress has been made over the last years in understanding the Duffy blood group system and its complexity. The Duffy blood group antigen serves not only as blood group antigen, but also as a receptor for a family of proinflammatory cytokines termed chemokines, and as a receptor for Plasmodium vivax malaria parasites. The Duffy antigen has been termed the "Duffy Antigen Receptor for Chemokines" (DARC) or the Duffy chemokine receptor. DARC might playa role as a scanvenger 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 most amply distributed human malaria in the world [51]. Individuals with the Duffy-negative phenotype are resistant to P. vivax invasion, and the molecular mechanism that gives rise to the phenotype Fy(a - b - ) in black individuals has been associated with a point mutation - 33TC expressed in homozigosity in the FYB allele [5]. Despite P. vivax be widespread throughout the tropical and subtropical world, it is absent from West Africa, where more than 95% of the population is Duffy negative. Recently, this point mutation has been described in heterozigosity in the FYA allele in others malaria endemic regions [7,8], 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 clinical medicine because of transfusion incompatibilities and hemolytic disease of the newborn (HDN) [1]. It is the number 008 in the International Society of Blood Transfusion (ISBT) nomenclature. The product of Duffy (FY) gene is a glycoprotein (gp-Fy), which spans the plasma membrane seven times and has an extra cellular N-terminal domain and an intracellular C-terminal domain. The system consists of four alleles, five phenotypes, and five antigens. The Duffy locus is located at chromosome l-lq22-23 [2]. Initially, two major co-dominant antigens were described, and designated Fya and Fyb, and codified by two co-dominant alleles, designated FYA and FYB [3]. Among Caucasians the anti-Fya and anti-Fyb antibodies 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 and FYB/FYB, respectively. The phenotype Fy(a - b - ) is more common among black individuals (Afro Americans or Occidental Africans) [4] The phenotype Fy(a - b - ) represents the genotype FYB SE/FYB SE (SE-silent erythroid), being FYB SE, a silent allele at locus FYA/FYB (Duffy). The FYB SE gene in the majority of black individuals that present the phenotype Fy(a - b - ), is an allele that is the same in its structural region as FYB, but has a point mutation in the GATA box promoter region, were the erythroid GATA-l transcription factor adheres, changing the transcription activity of this factor, abolishing the gene transcription in erythrocytes of individuals that present homozygosity for this mutation. It is common among black individuals but rare among other races [5]. In 1965, the FYX, or FYB wx (WK- Weak) allele at Duffy locus was described. The gene itself does not encode the production of a distinct antigen from others 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 Paulo, SP 04023-902, Brazil. Tel: 5511 5579 1550. Fax: 5511 5571 8806. E-mail: jobordin@hemato.epm.br
ISSN 1024-5332 printlISSN 1607-8454 DOl: 10.1080/10245330500469841 online 2006 Informa UK Ltd.

390 D. M. Langhi & J. O. Bordin

the FYB WK gene, have erythrocytes that react weakly with some, but not with other examples of anti-Fyb antibodies. However, the Fybwk antigen acts as a Fyb antigen of low expression, and there is no anti-Fybwk [6]. Two mutations in the coding region of the FYB gene are associated with low expression of this allele. Most of the time, Fybwk has been described among Caucasian individuals, where its occurrence is not rare. Recently, Zimmerman et al. [7] described the presence of the FYA NULL gene in a population of a malarial endemic area at Papua-New Guinea, and we have described the FYA NUL in a population of the Amazon region in Brazil [8]. Another function of the Duffy antigen is being a receptor for the Plasmodium vivax parasite of human malaria [9]. E vivax is practically absent in Occidental Africa, where more than 95% of the population is Duffy negative. The E vivax and the Plasmodium knowlesi need the interaction with the Duffy antigen to internalize within the erythrocyte [10,11]. The Duffy antigen also acts as a receptor for a family of pro-inflammatory cytokines, called chemokines, and is also called Duffy Antigen Receptor for Chemokines (DARC) [12]. Introduction The Fya antigen was identified serologically in 1950 by Cutbush et al. [3] during an investigation of a transfusion reaction in the serum of a multiply transfused patient with hemophilia. Anti-Fyb was discovered the following year by Ikin et al. [13]. Initially, the antigen with which the serum from the first patient reacted was named Fya and later the determinant gene of this antigen was called FYA. On this occasion, the existence of the allele FYB was postulated and was later demonstrated by Ikin et al. [13]. In Caucasians, these antibodies 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 and FYB/FYB, respectively. In 1955, Sanger et al. [4] reported that the phenotype Fy(a - b -) is more common in American Black individuals than is any phenotype in which Fya or Fyb is present. It was thought that the Fy(a - b - ) phenotype probably represented the FYB SE/FYB SE genotype, with FYB SE being a silent allele at the FYA/FYB (Duffy) locus. In blood transfusion, Fya and Fyb are the most important antigens in the Duffy system, and are fully developed at birth [14]. Anti-Fya is not a particularly uncommon antibody and is found both as a single entity in the sera of persons phenotypically Fy(a - b + ) and as one of a mixture of antibodies made by good responders. Anti-Fya is usually an IgG antibody, mostly IgG 1, most often immune, reacts optimally or only by IAT, and naturally occurring anti-Fya is rare [15]. Anti-Fya has been incriminated in immediate and delayed hemolytic transfusion reactions [16,17], and many cases of hemolytic disease of the newborn (HDN) due to anti-Fya, varying from mild to fatal, have been observed [18]. Anti-Fyb is found twenty times less frequently than anti-Fya [19], and most examples appear to occur in individuals who make multiple antibodies after exposure to red cells. Contreras et al. [20] have described the production of anti-Fyb by a woman exposed to the antigen when the fetus she was carrying was given an intrauterine transfusion, and Issit [21] has described a potent anti-Fyb in the serum of a nontransfused male donor with no known exposure to foreign red cells. Thus, the antibody appeared to be naturally-occurring. The Fy3 antigen was described in 1971 [22], and Fy4 and Fy5 in 1973 [23]. In 1987 the Fy6 antigen was recognized by a murine monoclonal antibody, and added to the Duffy system [10]. In 1965, Chown et al. [6] described a new allele, named FYX or FYB WK, as its product was totally uncertain, at the Duffy locus. The Fybwk antigen behaves as a weak Fyb, and there is no anti_Fybwk. It is inherited as an allele of FYA and FYB, co-dominant with FYA and recessive to FYB. This new allele was considered the fourth Duffy allele [6]. The gene does not encode production of an antigen that is distinct from others in the Duffy system. Instead, individuals who have inherited an FYB WK but not a FYB gene have red cells that react poorly with some, and not at all with other, examples of anti-Fyb and sometimes the antigen can be detected only by adsorption and elution of anti-Fyb [6]. The presence of Fybwk is associated with diminished expression ofFy3, Fy5 and Fy6 antigens, markedly in individuals homozygous for FYB WK, bind weakly to anti-Fy3, and anti-Fy6 [24]. The low expression of Duffy antigens in these cases is due to a low copy number of the Duffy protein in the cell surface, with approximately one-tenth as much expression, and not due to the conformational changes in the protein [25]. The Duffy system is number 008 in the ISBT nomenclature. Duffy protein The glycoprotein (gp-Fy) that expresses the Duffy antigens was initially described in 1982 by Moore et al. [26], and in 1984 Hadley et al. [27] demonstrated by immunodetection technique that the red blood cell (REC) component that carries the Duffy antigens is a protein with a molecular weight of 35-43 kDa, sensitive to chymotrypsin and resistant to trypsin treatments. The hydropathy map predicts an extracellular N-terminal domain of 60 residues, seven

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Duffy blood group and malaria

transmembrane a-helices, short protruding hydrophilic loops, and an intracellular C-terminal domain of 28 residues [28]. The gp- Fy is composed of the Fya and Fyb epitopes, in addition to the Fy3, Fy4, Fy5 and Fy6 epitopes. The Fya e Fyb epitopes are located in the extracellular domain, in resides 42 and the Fy3 epitope is located on the third extracellular loop. The Fy6 epitope has been accurately mapped to a heptapeptide comprising residues 19-25 and is located between the two glycosylation sites [29]. The Duffy protein is encoded by a short single copy gene (1.5 kb, 2 exons) located on chromosome 1 and exhibits significant protein sequence homology with the human and rabbit IL-8 receptors. DARC is most probably organized in seven transmembrane domains, similar to other members of the G-protein coupled chemokine receptors [30]. The major (336 aa) and minor (338 aa) DARC polypeptide isoforms are encoded by the spliced and unspliced Fy mRNAs, respectively, which differ by the sequence of the six and eight NHTterminal amino acids, respectively, but bind anti-Fy antibodies and chemokines equally well. DARC has a specialized tissue distribution as present on RBCs, endothelial cells of post-capillary venules (a site of leukocyte trafficking), Purkinje cells of the cerebellum and presumably on some epithelial cells in the kidney [31,32].

391

Duffy polymorphism The molecular basis of the Duffy blood group polymorphisms has been determined. The Duffy (FY) gene is located in the 1q22-q23 region of chromosome 1 [33]. The Duffy cDNA was originally cloned from a non-spliced mRNA, and it was assumed that Duffy is an intron less gene [34]. The Duffy transcription unit encompasses 1572 nt, including exon 1 of 55 nt, a single intron of 479 nt, and exon 2 of 1038 nt [35].
Exon 1
Duffy Gene 5'

The Fya/b antigenic polymorphism results from a point mutation at nucleotide 125, in the gp-FY gene, with the change of a single base G 125A. This change results in a single amino acid difference, changing glycine by aspartic acid at position 42 of gp-Fy. Glycine at position 42 (Gly42) defines the Fya antigen, and aspartic acid at position 42 (Asp42) defines the Fyb antigen [34] (Figure 1). The molecular mechanism that gives rise to the phenotype Fy(a - b - ) in black individuals has been classically associated with a point mutation - 33TC in the promoter region of a FY* B allele that disrupts a binding site for the transcription factor GATA-1, a mutation that specifically abolishes the transcriptional activity in erythroid cells GATA box of individuals that express this mutation in homozygosity [5] (Figure 2). This mutation defines the FYB SE allele, that is structurally identical to the FYB allele in its codifying region. Individuals showing heterozygosity for the GATA box mutation demonstrate dosage effect, and have approximately one-half the level of Duffy antigen in erythrocytes [25]. In Caucasians, the Fy(a - b -) phenotype is extremely rare and in all cases studied so far it is associated with mutations or deletions within the coding sequence of the DARe gene [32]. It is assumed that these mutations, in contrast to the erythroid specific mutation - 33TC, should lead to the true F~ull phenotype with a lack of expression ofDARC in both erythroid and nonerythroid cells. For individuals from malarial endemic regions in southeastern Asia, the Fy(a - b -) phenotype is practically nonexistent and the molecular basis of this phenotype is not the same as that described in Black individuals, in other words, homozygosity for the FYB ES allele [37]. In some locations, individuals having the Fy(a - b - ) phenotype possess the FYA/FYA and FYA/FYB genotypes, without the presence of the FYBES/FYBES genotype [37], therefore, there is heterogeneity in the findings. In this group of
Exon 2

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1572 (nts)

,
,------,
Promoter region

,,,,
"
" '-

3'

mRNA

5' ,

gp-Fy

NHZ

42

COOH
(residue number)

Fya Fyb

Gly Asp

Figure 1. Schematic representations of the Duffy gene FY (top), mRNA (middle), and gp-Fy (bottom), with polymorphism FYA/FYB at nt 125, and residues Gly for FYA and Asp for FYB at position 42 (Modified from Pogo, Chaudhuri [32]).

392 D. M. Langhi & J. O. Bordin

Point mutation nt-33T -7 C

FYA or FYB Gene 3'

\
\

Coding Region
\ \ \
, J

1008

FYBSE Gene

GATA-l Figure 2. Schematic representation of point mutation in GATA-I site, responsible for the non-expression of gp-Fy in RBCs of black individuals 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 the majority of Black individuals with the Fy(a - b - ) phenotype (Modified from Issit, Anstee [36]).

individuals a study was carried out on the phenotyping and genotyping and the presence of the antigen Fyawk with low reactivity to anti-Fya was observed. The presence of the Fya antigen which is weakly reactive was also described in Malaysians [38]. Recently, Zimmerman et al. [7] described the presence of this mutation linked to the FYA allele in a population living in a malaria endemic region of Papua New Guinea and this FYA null appears to have a more recent origin than that of FYB null. We demonstrated the presence of the FYA null allele in blood donors and Individuals from a malarial endemic region of Brazil [8]. The Fybwk phenotype is associated with a missense mutation in the coding region of FYB gene, with a single substitution at nt 265C- T that produces an amino acid change Arg89Cys in gp-Fy. Near this mutation, another mutation, G298A, resulting in AlalOOThr amino acid substitution was also identified. This mutation is silent and is also present in FYB or FYA alleles from Fy(a - b +) and Fy(a + b + ) individuals [25]. These amino acid substitutions occur in the first intracellular loop of gp-Fy, and only the Arg89Cys substitution produces the Fy(a - b + wk) phenotype (Figure 3). The amino acid substitutions occur in the first intracellular loop of gp-Fy, representing considerable

modification in the chemical nature of the site, and results in very low membrane expression of DARC in Fy(a - bweak) erythrocytes, entailing a weakening of the Fyb antigen [34]. The expression of Fy3, Fy6, and Fyb antigens, when the G298A mutation is present, is similar to those encoded by cDNA of FYB, contrary to the situation when the mutation C265T exists [25]. In non-Ashkenazi Jews, individuals with the phenotype Fy(a - b -) show the promoter wildtype GATA, however, Non-Ashkenazi Jewish individuals, with the Fy(a - b -) phenotype, were described as presenting the region which promotes the FYB gene in wild type form, but with the C265T mutation in the codifying region of the FY gene, which alters the antigenic determinants of the DARC, weakening the Fyb antigen [39]. Also described were non-Ashkenazi Jewish individuals, with the Fy(a - b -) phenotype and the FYB/FYB genotype, heterozygotes for the GATA mutation, entailing simultaneously the C265T mutation, weakener of FYB [39]. Similar to that which was described in nonAshkenazi Jews, was also described in Brazilian Black individuals, namely the association of the mutation responsible for the FYB fraco genotype (C265T) with the T-33C mutation (FYB es) in the

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Exon 1 Duffy Gene 5' /' ,------,' Promoter region mRNA ", " ", 1: .

Exon 2

:
1008 (nts)
I

5' ,

-3'AAAn

gp-Fy

NH
2

100
89
Arg/Cys

(residue number)

Alaffhr

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

and the

Duffy blood group and malaria individuals whose Fy(b -) phenotype cannot be explained by the isolated mutation in the GATA-1 "box" [40].

393

Functional aspects Although all blood group antigens are serologically detectable on RBCs, most of them are also expressed in non-erythroid tissues, raising further questions on their physiological function under normal and pathological conditions. In addition to their structural diversity, blood group antigens also possess wide functional diversity, and can be schematically subdivided into five classes: (a) transporters and channels; (b) receptors for ligands, viruses, bacteria and parasites; (c) adhesion molecules; (d) enzymes; and (e) structural proteins. Many RBCs surface molecules among those which carry blood groups are receptors for viruses, bacteria, and parasites suggesting that these antigens may playa direct role in the pathogenesis of infectious diseases [41,42]. A variety of microorganisms recognize carbohydrate structures present on glycolipids and glycoproteins, for instance sialic acids, which are abundantly represented on glycophorin A (GPA), or the Cala 1-4 Gal motif shared by P, Pk and PI glycolipids used by several bacterial strains and toxins responsible for upper urinary tract infections [43]. The Duffy antigens (Fya/b, Fy3 and Fy6) are carried by a membrane glycoprotein exhibiting two interesting biological properties: as a promiscuous receptor for chemokines of the CC (RANTES, MCP-1) and CXC (IL8, mgSA) subfamilies of proinflammatory peptides named according to the structure of a conserved cysteine (C) motif. Hence, the Duffy protein was renamed DARC for "Duffy Antigen/Receptor for Chemokines"; as erythroid receptor for R vivax and R knowlesi.

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CXC) also bind to the Duffy blood group glycoprotein, which is now called DARC. Of the cellular chemokine receptors, CXCR4, CCR5 and Duffy are the only receptors that have been unequivocally proven to act as coreceptors for cell entry of pathogens: Duffy for the entry of malarial parasite, R vivax, and CCR5 for the entry of M-tropic strains of human immunodeficiency virus (HIV) [47]. During experiments with chemokine receptors in erythrocytes, the perfect correlation between the bonding of the chemokines to the erythrocytes and the presence of the Duffy antigen was well established [12]. It has been demonstrated that anti-Fy antibodies inhibited the chemokine binding ability to Duffy positive erythrocytes, and chemokines that bind to erythrocytes block RBC invasion by malarial parasites, that use the Duffy antigen as a receptor [12]. CC and CXC chemokines bind to DARC with high affinity, suggesting some role in inflammatory reactions [31]. DARC might play a role as a scavenger on the RBC surface to eliminate excess of toxic chemokines produced in some pathologic situations [44]. There are some experimental studies suggesting that DARC is a redundant protein that may playa role in the regulation of induced leukocyte trafficking in vivo [48]. The CXCR4 and CCR5 chemokine receptors are mandatory cofactors for HIV infection, by interacting with the viral envelope gp 120 in the presence of CD4 [49]. A report shows that HIV-1 viral particles might bind to the Fy protein, suggesting that RBCs may function as a virus reservoir or as a receptor for the entry in some cells [50]. Duffy antigen receptor for R vivax R vivax causes approximately between 70 and 80 million cases of malaria per year and is the most amply distributed human malaria in the world [51]. One of the most interesting aspects of the Duffy antigen is its function as a receptor for the human malaria parasite R vivax. In 1975, Miller et al. [9] showed that Duffynegative human RBCs were resistant to invasion by R knowlesi, a monkey malaria parasite that was known to be capable of invading human RBCs and, in rare instances, of infecting humans. Furthermore, anti-Fya and anti-Fyb blocked invasion of R knowlesi into Fy(a + b -) and Fy(a - b +) RBCs, respectively, and the treatment of the RBCs with enzymes which remove from their surface the antigenic determinants Fya e Fyb (chemotrypsin and pronase), making the RBCs resistant to invasion [9]. Studies on R knowlesi were extended to R vivax, a human malaria that is second only to Plasmodium faleiparum in terms of the toll it takes on populations of endemic areas. Although, R vivax is widespread throughout the tropical and subtropical world and

Duffy antigen receptor for chemokine The fact that RBCs possess a chemokine receptor was first established by Darbone et al. [44]. Chemokines constitute a family of proinflammatory cytokines capable of activating leukocytes and causing chemotaxis, but other important functions have been discovered, including angiogenic and angiostatic activities [45]. The number and spacing of aminoterminal cysteines have been used in the classification of chemokines into four families C, CC, CXC, and CXXXC. The biological effect of chemokines is mediated by the binding and activation of G-proteincoupled, seven-transmembrane domain chemokine receptors [46]. Some chemokines (classes CC and

394 D. M. Langhi & J. O. Bordin

is a major drain on the health care resources of India and Southeast Asia, it is absent from West Africa, where more than 95% of the population is Duffy

R vivax merozoites invade reticulocytes that express the Duffy protein and the counter receptor is a 140 kDa protein (PvDBP) that belongs to Plasmodium adhesion proteins, including EBA-175 and PfEMPl
(see above), all having in the NHz-ter region a cysteine-rich domain (called DPP region II) mediating erythrocyte binding [58,59]. It is not known whether the Duffy antigen is a structural junction component because the molecular nature of junction has yet to be determined. It is clear, however, that the antigen interaction of the parasite Duffy-receptor is crucial to the formation of the junction and subsequent invasion [57]. Several blood group molecules of red cell surface contribute to the complex mechanisms of invasion and sequestration steps that characterize the most severe form of malaria caused by R jalciparum. R jalciparum merozoites may invade RBCs through sialic aciddependent and independent pathways [60,61]. GPA is involved in the sialic-acid dependent pathway and the counter receptor of GPA is the parasite protein erythrocyte binding antigen (EBA) of 175 kDa [62], which binds to sialic acid and peptide backbone ofGPA through a cysteine-rich domain (region II) analogous to the one mediating R vivax/Duffy protein interaction [63]. Recently, a homologue to EBA-175 called BAEBL has been shown to use GPC for invasion [64]. Rosette formation and cytoadherence of R jalciparum-infected erythrocytes to vascular endothelium result in the sequestration of infected cells particularly in the brain vasculature (resulting in anoxia, altered brain function and coma), and thus represent a major cause of cerebral malaria [65]. However, the role of the immune system is likely also to be of critical importance [66]. Cytoadherence ofRBCs containing mature-stage parasites to vascular endothelium and platelets is a protective mechanism used by the parasite to avoid elimination. This is mediated by several membrane proteins receptors present on endothelial cells (CD36, ICAM-l, PECAM-l, TSP, chondroitin sulfate), and cryptic antigens ofRBCs like those exposed on altered Band 3 [67]. The parasitized RBCs not only adhere to vascular endothelium but also adhere to uninfected RBCs, a process known as "rosette" formation. This is mainly mediated by the complement receptor CRI (CD35, carrier of Knopps antigens), since among a large series of null variant erythrocytes tested for the ability to form rosettes, only those of the Helgenson phenotype (CRI-deficient) were negative. In addition Sla( - ), red cells, which have a reduced copy number of CRl, rosetted poorly [68]. The counter receptor of CRI is PfEMP 1 (R jalciparum Erythrocyte Membrane Protein 1), a protein of 300350 kDa produced by a gene of the varfamily, but other ligands of PfEMP 1 (heparin sulfate glycans, ABO groups, CD36) are also involved in rosette formation [69]. The main function of CRI (190-280 kDa) is to capture and remove from the liver and spleen immune

negative. Miller et al. [52] showed that resistance to R vivax correlates with the Duffy-negative phenotype, and more recently, Barnwell et al. [53] showed that R vivax merozoites are incapable of invading Duffy-negative RBCs. Thus, R vivax, like R knowlesi, relies on a Duffy antigen-parasite ligand interactions for invasion. It should be noted that R vivax preferentially invades reticulocytes [54] although mature RBCs as well as reticulocytes express a coreceptor that, along with the Duffy antigen, is necessary for optimal invasion by R vivax. The bonding agent of the R vivax parasite, which specifically bonds itself to the reticulocytes, was also identified and the cDNA encoding this protein was cloned [54]. When the anti-Fy6 antibody became available, studies demonstrated that the presence of Fy6 is what results from the invasion of human RBCs by the merozoites of the R vivax [53]. In humans, the presence or absence of Fy6 correlates with the presence or absence of the Fya and Fyb. In other words, the Fy(a +) or Fy(b + ) RBCs are always Fy6 positive and Fy(a - b - ) RBCs are Fy6 negative. The invasion of the Fy(a + ) RBCs by R vivax can be partially blocked by the covering of the RBC with anti-Fya [9], and totally blocked by covering the RBC with anti-Fy6 [55]. The fact that the great majority of black African and Afro-American individuals are resistant to infection by R vivax is directly related to the Fy(a - b - ) phenotype, or in other words, the absence of the Fya and Fyb antigens. One Duffy binding protein of R vivax, called "PvDAP-l" (Pv - indicates R vivax and DAP indicates Duffy-associating protein) has a critical role as a bonder in the adhesion of the parasite and posterior invasion of the positive Duffy RBCs [56]. R vivax, as with other species of human Plasmodium, initiate erythrocyte invasion through the expression of various surface organelles and structures on the merozoite which bind with the surface proteins of the erythrocyte. The well-characterized interaction of the bonder-receptor involves the Duffy bonding protein, expressed in the merozoite form of the R vivax and its corresponding receptor in the erythrocyte, the receptor DARC. This interaction is unique among human malaria infections, where the interaction of the bonding receptor is essential to the invasion of the erythrocyte by R vivax. Alternate models of erythrocytic invasion have not yet been described [57]. This Duffy bonding protein, of R vivax, belongs to the family of bonding proteins for erythrocytes, which also includes the bonding protein for sialic acid R jalciparum and to the Duffy bonding protein R knowlesi.

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Duffy bloodgroup and malaria

complexes containing C3b and C4b, but it also plays a role in the immune response [70]. The CR1 level on RBCs is low but may vary widely. The extra membranous region of CR1 is made of 30 short consensus repeats (SCR) and two Knopps blood group polymorphisms have been assigned to the homologous region D within SCR24 (McCa/b = K1590E) and SCR25 (SlaNil = R160 1G) [71]. Duffy polymorphism and malaria

395

Malaria is an important selective force for human genetic adaptations due to the sustained, lethal impact that it has had on human populations around the world. Homozygosity for the Duffy-negative blood group antigen confers complete resistance to vivax malaria. Nevertheless, it is unclear whether selective pressure of vivax malaria alone was the main cause for the emergence and fixation of the FYB null/ FYB null genotype in much of West Africa where vivax malaria is absent. Indeed there is little doubt that malaria caused by E vivax is not as directly lethal as E jalciparum, but a fulminant E vivax infection still causes serious morbidity in those living at the minimum subsistence level. The emergence of the new FY*A allele carrying the - 33TC mutation in E vivax-endemic region of Papua New Guinea, further supported the hypothesis that E vivax malaria can act as a selective agent of the Fy(a - b - ) phenotypes in this region [7]. Accordingly, Fy mRNA and DARC are normally expressed in nonerythroid tissues in populations of West Mrica, where E vivax is no longer present. It is assumed that the most common phenotype Fy(a - b - ) represents an adaptive response to resist malarial infection [32,5]. RBCs from individuals homozygous for the wildtype promoter (Genotypes FYA/FYA, FYB/FYB and FYA/FYB) express twice the amount of Fy antigen than those heterozygous for the GATA-1 mutation (genotypes FYA/FYB SE, FYB/FYB SE) [72,7], but the biological significance of this finding is unknown [7]. In individuals who present the GATA mutation in the heterozygous form, dose effect has been demonstrated, being that only 50% of the Duffy antigens are expressed in the erythrocytes [73]. These data suggest that heterozygosity for the - 33TC mutation in the allele promotes the protection against E vivax infection, but is still susceptible to it [74]. When we correlated the frequency of - 33TC mutation and the development of malaria by E vivax, in individuals from a malarial endemic region in Brazil, the data suggest that the - 33TC mutation in heterozygotes does not give protection against E vivax infection [8]. Another study among patients infected by E vivax in Brazil, showed that the proportion of individuals that did not present the - 33TC mutation in homozygotes

was similar among those infected by E vivax and non-E vivax. If it were the case that the mutation in heterozygous form conferred any degree of protection against E vivax infection, one would expect to encounter a relative excess of patients without the mutation among individuals infected by E vivax [75]. In vitro binding assays have proved a significant decline in binding activity with heterozygous Duffypositive/negative genotypes, both FYA/FYA null and FYB/FYBnull, when compared to the homozygous Duffy-positive erythrocytes. In these assays, cytoadherence between the DBP ligand domain and erythrocytes from Duffy promoter heterozygous donors was significantly reduced [74]. Previous studies have confirmed that heterozygous Duffynegative individuals remain susceptible to infections by E vivax [76], studies made by Michon et al. [74] suggested that even at the heterozygous state the Duffy-negative allele can confer a quantifiable resistance advantage against E vivax. In all 23 individuals from Papua New Guinea found with the FYA null allele, all were heterozygous (FYA/FYA null) for the allele. Flow cytometric analysis revealed that these individuals expressed half the amount of Duffy antigen on their erythrocytes indicating a gene dosage effect. A definitive conclusion could not be made on whether these heterozygous individuals were less susceptible to infection with E vivax [74]. However, it has been demonstrated that PvDBP adherence to RBCs is significantly reduced for erythrocytes from heterozygous individuals carrying this mutation, suggesting that this new allelic form of Duffy negativity is correlated with resistance against E vivax malaria [74]. Other studies demonstrated the presence of RBCs which react weakly with anti-Fya in eight individuals of Thai ethnicity who live in a region endemic to malaria [77]. These phenotypes may occur due to the decrease in the number of RBC antigens, to structural alteration of the antigen itself, or even to both motifs [77]. This weak Fya antigen could, in a manner similar to that observed in Africa, offer a selective advantage to this population in relation to malaria, as the Fya antigen is predominant in the region. Among Indonesians who live in regions endemic to malaria, a study was carried out on the phenotyping and genotyping and the presence of the antigen Fya with low reactivity to the anti-Fya was observed, also occurring a discrepancy between the genotype and phenotype of these individuals, suggesting another genetic alternative for the individuals presenting Fy(a - b - ) different from the classic genetic base for this phenotype in Mricans [37]. The presence of the Fya antigen which is weakly reactive was also described in Malaysians [38]. In our study, 5 individuals from malaria endemic region in the Amazon, presented the FYA null allele. Four individuals presented the Fy(a + b - )

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D. M. Langhi & J 0. Bardin (FYA/FYA null) for the allele. Flow cytometric analysis revealed that these individuals expressed half the amount of Duffy antigen on their erythrocytes indicating a gene dosage effect. A definitive conclusion could not be made on whether these heterozygous individuals were less susceptible to infection with F?vivax [74]. However, it has been demonstrated that PvDBP adherence to RBCs is significantly reduced for erythrocytes in heterozygous individuals carrying this mutation, suggesting that this new allelic form of Duffy negativity is correlated with resistance against F?vivax malaria [74]. In our study, 5 individuals from malaria endemic region in the Amazon, presented the FYA null allele. Four individuals presented the Fy(a + b - ) phenotype, the FYA/FYB genotype and the - 33TC mutation in the homozygous form, and one individual presented the Fy(a + b - ) phenotype, the FYA/FYA genotype and the - 33TC mutation in the heterozygous form. Of the four individuals with the Fy(a + b -) phenotype and FYA/FYB genotype, two never acquired malaria, one acquired malaria by F?faleiparum and one did not inform his epidemiological status for malaria. The individual who presented the Fy(a + b - ) phenotype and FYA/FYA genotype, had acquired malaria by F?vivax and F? faleiparum [8]. Therefore, due to the small number of cases, we cannot affirm that the presence of the - 33TC mutation in the FYA allele, isolated or in association with the mutation in the FYB allele, can confer a certain degree of protection against infection by

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phenotype, the FYA/FYB genotype and the - 33TC mutation in the homozygous form, and one individual presented the Fy(a + b - ) phenotype, the FYA/FYA genotype and the - 33TC mutation in the heterozygous form. Of the four individuals with the Fy(a + b -) phenotype and FYA/FYB genotype, two never acquired malaria, one acquired malaria by F?faleiparum and one did not inform his epidemiological status for malaria. The individual who presented the Fy(a + b - ) phenotype and FYA/FYA genotype, had acquired malaria by F?vivax and F?faleiparum [8]. Therefore, due to the small number of cases, we cannot affirm that the presence of the - 33TC mutation in the FYA allele, isolated or in association with the FYB allele, can confer a certain degree of protection against infection by F?vivax, despite being demonstrated by Michon et al. [74] a smaller expression of the Duffy antigens in the RBCs of individuals with the - 33TC mutation, and that situation could be exacerbated by the presence of the mutation in both FYA and FYB allele. No data on F? vivax susceptibility are currently available for individuals that express the FYB weak allele. Summary Since the molecular basis of the Duffy blood group polymorphisms has been determined, the molecular mechanism that gives rise to the phenotype Fy(a - b - ) in black individuals has been classically associated with a point mutation - 33TC in the promoter region of a FY* B allele, a mutation that when present in homozygosity confers protection against F?vivax infection [5]. The described FYA null appears to have a more recent origin than that of FYB null, and this allele has been reported to occur in New Guinea [7] and Brazil [8]. RBCs from individuals homozygous for the - 33TC mutation express twice the amount of Fy antigen than those heterozygous for the mutation, but the biological significance of this finding is unknown [72,7] . In individuals who present the GATA mutation in the heterozygous form, dose effect of Duffy antigens has been demonstrated [25], and these data suggest that heterozygosity for the - 33TC mutation promotes protection against F? vivax infection, but remains susceptible to it [74]. When we correlated the frequency of the - 33TC mutation and the development of malaria by F?vivax, in individuals from a malarial endemic region in Brazil, the data suggest that the - 33TC mutation in heterozygous individuals does not give protection against F?vivax infection [8]. In individuals from Papua New Guinea found to have the FYA null allele, all of them were heterozygous

F?vivax.
No data on F? vivax susceptibility are currently available for individuals that express the FYB weak allele.

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