Hematology

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Duffy blood group and malaria

Dante M. Langhi & José Orlando Bordin

To cite this article: Dante M. Langhi & José Orlando Bordin (2006) Duffy blood group and malaria,
Hematology, 11:5-6, 389-398, DOI: 10.1080/10245330500469841

To link to this article: https://doi.org/10.1080/10245330500469841

Published online: 04 Sep 2013.

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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)

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 represent the genotypes FYA/ FYA, FYA/ FYB and

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
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 online © 2006 Informa UK Ltd.

DOl: 10.1080/10245330500469841
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 pheno-
types 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 non-
transfused 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 extra-
cellular N-terminal domain of 60 residues, seven

transmembrane a-helices, short protruding hydro-
philic 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].
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 5'
Gene , ,,,,
,------, "
Promoter "
region
mRNA
gp-Fy
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]).
Duffy blood group and malaria 391
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 1572 (nts)
3'
'-
,
5' ,
NHZ COOH
Fya
•
42
Gly
(residue number)
Fyb Asp
392 D. M. Langhi & J. O. Bordin

Point mutation
nt-33T -7 C

FYA or FYB Gene

3'
\
\ Coding Region 1008
\
FYBSE Gene \
\ I
, J

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 modification in the chemical nature of the site, and
and genotyping and the presence of the antigen Fyawk results in very low membrane expression of DARC in
with low reactivity to anti-Fya was observed. Fy(a - bweak) erythrocytes, entailing a weakening of
The presence of the Fya antigen which is weakly the Fyb antigen [34].
reactive was also described in Malaysians [38]. The expression of Fy3, Fy6, and Fyb antigens, when
Recently, Zimmerman et al. [7] described the the G298A mutation is present, is similar to those
presence of this mutation linked to the FYA allele in a encoded by cDNA of FYB, contrary to the situation
population living in a malaria endemic region of Papua when the mutation C265T exists [25].
New Guinea and this FYA null appears to have a more In non-Ashkenazi Jews, individuals with the
recent origin than that of FYB null. phenotype Fy(a - b -) show the promoter wild-
We demonstrated the presence of the FYA null allele type GATA, however, Non-Ashkenazi Jewish indivi-
in blood donors and Individuals from a malarial duals, with the Fy(a - b -) phenotype, were
endemic region of Brazil [8]. described as presenting the region which promotes
The Fybwk phenotype is associated with a missense the FYB gene in wild type form, but with the C265T
mutation in the coding region of FYB gene, with a mutation in the codifying region of the FY gene, which
single substitution at nt 265C- T that produces an alters the antigenic determinants of the DARC,
amino acid change Arg89Cys in gp-Fy. Near this weakening the Fyb antigen [39].
mutation, another mutation, G298A, resulting in Also described were non-Ashkenazi Jewish indivi-
AlalOOThr amino acid substitution was also ident- duals, with the Fy(a - b -) phenotype and the
ified. This mutation is silent and is also present in FYB FYB/FYB genotype, heterozygotes for the GATA
or FYA alleles from Fy(a - b +) and Fy(a + b + ) mutation, entailing simultaneously the C265T
individuals [25]. These amino acid substitutions occur mutation, weakener of FYB [39].
in the first intracellular loop of gp-Fy, and only the Similar to that which was described in non-
Arg89Cys substitution produces the Fy(a - b + wk) Ashkenazi Jews, was also described in Brazilian
phenotype (Figure 3). Black individuals, namely the association of the
The amino acid substitutions occur in the first mutation responsible for the FYB fraco genotype
intracellular loop of gp-Fy, representing considerable (C265T) with the T-33C mutation (FYB es) in the

Exon 1 Exon 2
Duffy 5'
Gene /'
,------,' ", :
Promoter " 1: 1008 (nts)
region ", .••
•••
I

mRNA 5' , -3'AAAn

gp-Fy NH
2
•• ••
89 100 (residue number)

Arg/Cys Alaffhr

Figure 3. Schematic representation of FY gene, with the C265T mutation (FYB WK), encoding the amino acid change Arg89Cys, and the
silent mutation G298A codifying the amino acid change AlalOOThr (Modified from Pogo, Chaudhuri [32]).

individuals whose Fy(b -) phenotype cannot be
explained by the isolated mutation in the GATA-1
"box" [40].
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 sub-
divided 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 Chemo-
kines";
• as erythroid receptor for R vivax and R knowlesi.
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 amino-
terminal 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-protein-
coupled, seven-transmembrane domain chemokine
receptors [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, 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 Duffy-
negative 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
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
negative. Miller et al. [52] showed that resistance to
R vivax correlates with the Duffy-negative pheno-
type, 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.
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) mediat-
ing 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 acid-
dependent 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 endo-
thelium 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 endo-
thelium 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 300-
350 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

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
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 wild-
type 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 demon-
strated, 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
Duffy bloodgroup and malaria 395
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 Duffy-
positive/negative genotypes, both FYA/FYA null and
FYB/FYBnull, when compared to the homozygous
Duffy-positive erythrocytes. In these assays, cytoad-
herence between the DBP ligand domain and
erythrocytes from Duffy promoter heterozygous
donors was significantly reduced [74]. Previous
studies have confirmed that heterozygous Duffy-
negative 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 - )
396 D. M. Langhi & J 0. Bardin
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 hetero-
zygous 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 epidemiolo-
gical 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 pro-
motes 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
(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 - ) pheno-
type, 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 hetero-
zygous 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 epidemiolo-
gical 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
F?vivax.
No data on F? vivax susceptibility are currently
available for individuals that express the FYB weak
allele.
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