Do FY antigens act as minor histocompatibility antigens in the graft-versus-host disease paradigm after human leukocyte antigen-identical sibling hematopoietic stem cell transplantation?

Abstract

FY antigens are candidate minor histocompatibility antigens relevant to renal allograft rejection, but no data have been reported about their role in graft-versus-host disease (GVHD) incidence after human leukocyte antigen (HLA)-identical siblings hematopoietic stem cell transplantation (HSCT). The aim of this study was to examine the effect of donor/recipient disparity at FY antigens on the incidence of GVHD in Tunisian patients receiving an HLA-identical HSCT. This work enrolled 105 Tunisian pairs of recipients and their HLA-identical sibling donors of HSCs. FY genotyping was performed with the polymerase chain reaction-sequence-specific primer method and donor/recipient disparity for these antigens was analyzed at two levels: incompatibility and nonidentity. The case-control analyses showed no significant correlation between FY disparity and the incidence of either acute or chronic GVHD. Sample size calculation showed that 572 cases and 1716 controls would be necessary to be able to detect a significant association with 80% power and two-sided type I error level of 5% (α=0.05). The lack of association in the studied cohort may be explained by the low immunogenicity of FY antigens in HSCT context, compared with other antigens such as HA-1 and CD31.

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S C I E N T I F I C C O M M U N I C A T I O N S
Do FY Antigens Act as Minor Histocompatibility
Antigens in the Graft-Versus-Host Disease
Paradigm After Human Leukocyte Antigen-Identical
Sibling Hematopoietic Stem Cell Transplantation?
FY antigens are candidate minor histocompatibility antigens
relevant to renal allograft rejection, but no data have been re-
ported about their role in graft-versus-host disease (GVHD) in-
cidence after human leukocyte antigen (HLA)-identical sibling
hematopoietic stem cell transplantation (HSCT) . The aim of
this study was to examine the effect of donor/recipient disparity
at FY antigens on the incidence of GVHD in Tunisian patients
receiving an HLA-identical HSCT . This work enrolled 105 Tu-
nisian pairs of recipients and their HLA-identical sibling donors
of HSCs . FY genotyping was performed with the polymerase
chain reaction–sequence-specific primer method . The case-
control analyses showed no significant correlation between FY
disparity and the incidence of either acute or chronic GVHD .
The lack of association in the studied cohort may be explained
by the low immunogenicity of FY antigens in HSCT context,
compared with other antigens such as HA-1 and CD31 .
Introduction
The ability of allogeneic stem cell transplantation (hematopoi-
etic stem cell transplantation [HSCT]) to cure several hemato-
logical malignancies has been widely recognized .1 However, this
therapy is often complicated by severe graft-versus-host disease
(GVHD), which remains the major cause of mortality affect-
ing about 50% of all patients even in human leukocyte anti-
gen (HLA)-identical siblings .2,3 The pathogenesis of GVHD is
mediated by alloreactive T CD4+ and/or CD8+ cells directed
against the recipient’s minor histocompatibility antigens (Mi-
HAgs) that are not expressed in the donor .2-4 MiHAgs are a
variety of endogenously synthesized polymorphic products that
can be recognized by alloreactive T cells only in the context
of major histocompatibility complex molecules (HLA) .5-7 The
importance of autosomal and sex-linked MiHAgs in the GVHD
paradigm has been extensively described .2,4,6,8,9 The FY antigens
are candidate MiHAgs relevant to renal allograft rejection,10
but no data have been reported about their role in GVHD inci-
dence after HLA-identical sibling HSCT .
FY antigens belong to the Duffy blood group system that is of
major interest in therapeutic medicine .11 This serologically
determined system consists of two antithetic antigens, FY1
(Fya) and FY2 (Fyb), encoded by a single FY gene that maps
to chromosome 1q22–q23 .12 This gene contains four major al-
lelic forms: two codominant alleles FY*1 and FY*2 coding, re-
spectively, for the FY1 and FY2 antigens; FY*0 is the FY silent
version and is not expressed only at the erythroid level; and an
FY*X allele producing a very weak FY2 antigen . The molecular
bases of FY polymorphism have been well described .13,14
The Duffy molecule (gpD) is a transmembrane glycoprotein of
35–43 kDa expressed in two forms, α and β, of 338 and 336
amino acids, respectively . This molecule contains an NH2 ex-
tracellular domain containing 59 amino acids, which seems to
be very important at the histocompatibility level (a candidate
MiHAg), especially in transplantation contexts .10,15 In fact, this
peptide sequence contains the FY1/FY2, FY6, and a part of the
FY3 antigen, which are linked to certain hemolytic diseases
such as that affecting the newborn (HDN) .15,16 Among others,
Duffy molecule is expressed on RBCs, cerebellar neurons, epi-
thelial cells of nonerythroid organs, and endothelial cells lin-
ing postcapillary venules throughout the body, which represent
the first site of contact with the donor alloreactive T cells after
HSCT .17 Thus, we suspect that Duffy molecule polymorphism
may affect the occurrence of the GVHD reaction after HLA-
identical HSCT by acting as MiHAg . Therefore, we decided to
examine association in a Tunisian cohort of patients receiving
an HLA-identical sibling HSCT . For this purpose, we selected
the FY*1/FY*2 (rs12075) polymorphism coding for FY1/FY2
antigens for molecular analysis .
Materials and Methods
Informed consent was obtained from all participants and/or
their families to participate in this study according to a proto-
col approved by the ethical committee for scientific and medi-
cal research of the National Blood Transfusion Centre of Tunis
(Tunisia) .
Patient?Selection
The cohort contains 105 Tunisian pairs of patients and their
HLA-identical siblings . The patients have undergone the
HLA-matched HSCT in the National Bone Marrow Transplan-
tation Centre of Tunis between January 2000 and March 2010 .
All patients received cyclosporine A and/or methotrexate for
GVHD prophylaxis and had either grades 0–I/grades II–IV
acute GVHD or chronic GVHD . For other clinical character-
istics, see Figure 1 .
Mohamed Hichem Sellami, Manel Chaabane, Houda
Kaabi, Lamia Torjemane, Saloua Ladeb, Tarek Ben
Othmane, and Slama Hmida

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DNA?Samples
EDTA-treated blood samples were collected from all individu-
als before HSCT . DNA was purified from peripheral blood leu-
kocytes using the DNA blood minikit (Qiagen) according to
the manufacturer’s instructions .
GVHD?Grading
The diagnosis and grading of acute and chronic GVHD were
defined according to previously published standard clinical cri-
teria .18
DNA?Amplification?Using?Polymerase?Chain?Reaction–sequence-
specific?Primers?
Molecular amplification was performed as described in Sel-
lami .13
Statistical?Analysis
Donor/recipient disparity at FY antigens is defined as follows:
• Nonidentity: when the donor and the recipient are not carri-
ers of the same FY genotype .
• Incompatibility: when the donor is not compatible with the
recipient for the FY alleles .
Chi-square and Fisher’s exact tests were used to analyze the
association between both FY nonidentity/incompatibility and
either acute or chronic GVHD occurrence among the whole
cohort . The cohort was subdivided into subgroups according to
similarities of the peptide binding motif of their HLA class I; the
association between both FY non-identity/incompatibility and
either acute or chronic GVHD was tested with univariate anal-
yses . Subgroups for which p-values were < 0 .2 were reexamined
in multivariate logistic regression models including major risk
factors such as adulthood and conditioning . Odds ratios (OR)
and p-values for univariate and multivariate analyses were used
to analyze the correlation between risk factors and the prob-
ability of both acute and chronic GVHD . These analyses, mod-
elled according to the default method and Hosmer-Lemeshow
goodness-of-fit, were performed with SPSS software (V 17 .0) .
Positive correlation was considered only when p-value was <
0 .05 (type I error level or a) .
A sample size calculation was done using the Power and Sample
Size Program PS V3 .0 .43 (January 2009)19 to determine the
sample size needed to detect a significant association between
the donor/recipient FY disparity and the GVHD incidence in
the recipient of HSCs (i .e ., to reject the null hypothesis H0) .
For this purpose, we made the following assumptions: two-sided
type I error level (α) of 0 .05, a type II error level (β) of 0 .2
(power of 80%), and a control/case ratio of 30 .
Results
General?Case–Control?Analysis
In this part of the study, we performed a general association
analysis using donor/recipient FY disparity and incidence of
GVHD after HSCT . This analysis showed no significant cor-
relation between either FY nonidentity or incompatibility and
the incidence of acute and chronic GVHD among recipients of
HSCs (Figure 2) . Thus, we decided to subdivide the cohort into
subgroups according to similarities of the peptide binding motif
of their HLA class I and reexamine these associations separately
in each group .
Subgroup?Case–Control?Analyses
As there is a lack of a significant association in the general anal-
ysis, we subdivided suspected the cohort into HLA class I sub-
groups . Subgroups are as follows: A2 (donor/recipient pairs car-
rier of the HLA-A*02 except A*0207, A*6802, and A*6901
alleles), other than A2 (pairs carrier of HLA-A alleles other
than of the first group), B7-like (pairs carrier of HLA-B7, -B35,
-B51, -B53, -B54, -B55, -B56, -B67, B78 alleles), B44-like (pairs
carrier of HLA-B37, -B41, -B44, B45, -B47, -B49, -B50, -B60,
-B61 alleles), and other than B7-like/B44-like (donor/recipient
pairs carrier of HLA-B alleles other than of B7-like/B44-like
groups) . In this model also, we did not find any significant as-
Figure 1. Clinical characteristics of the patient/donor pairs enrolled
in the study (M, male; F, female; < or > 16, < or > 16 years; Y, ma-
lignant; N, nonmalignant; TBI, conditioning with TBI; other, other
conditioning regimens.
Figure 2. Analysis of association between both FY non-identity
and incompatibility and incidence of either acute or chronic GVHD
among patients enrolled in this study. GVHD, graft-versus-host
disease.

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sociation (Figures 3 and 4) . Consequently, we decided to re-
analyze only groups in which we observed a p-value of < 0 .2
in a multivariate context including major risk factors such as
adulthood and conditioning .
Multivariate?Analysis
This analysis considered only the relationship between FY1/
FY2 identity and chronic GVHD incidence in the HLA-B7-
like group (p = 0 .167, OR= 1 .031) . We analyzed the combined
effects of FY antigens identity, disease, donor/recipient sex mis-
match, adulthood, and conditioning, which were described as
potential risk factors (Figure 5) . As for the other analyses, in-
teractions between these factors were also not significant for
GVHD .
Discussion
The general case-control analysis showed no correlation be-
tween donor/recipient disparity at FY antigens and the inci-
dence of either acute or chronic GVHD among recipients of
HSCs . Even when subdivided into subgroups according to the
peptide binding motif of HLA class I there was no significant
correlation between FY and GVHD . The lack of association
between FY disparity and GVHD incidence was also observed
when we performed a multivariate analysis for the HLA-B7
group (p-value of < 0 .2) . In this model, we included more risk
factors such as donor/recipient sex mismatch and conditioning
with TBI, but it appeared that the combined effects of these fac-
tors have no influence on GVHD . Thus, it appears that donor/
recipient disparity at FY antigens is not critical in GVHD . This
finding may be explained by several lines of reasoning . First, we
believe that the absence of relevant correlation is due to the
low immunogenicity of the FY antigens compared with other
antigens such as HA-1 and CD31 . Further, there are no data
suggesting the involvement of FY antigens in T-cell–mediated
immune responses such as those in GVHD reaction . In fact,
immunogenicity of FY antigens has been usually described as
a potential cause of developing allo-antibodies .11 The contri-
bution of humoral immunity in the GVHD paradigm is poorly
defined, although Miklos et al . reported a correlation between
antibody responses to H-Y MiHAg and chronic GVHD (2005) .
Thus, examining the anti-FY level in sera of recipients who
were mismatched with their donors of HSCs at Duffy antigens
may be useful .
Likewise, the use of a CSA/MTX-based prophylaxis proto-
col by patients enrolled in this study may explain the lack of
a significant association . This prophylaxis is used to prevent
GVHD incidence after HSCT, and we believe that the real ef-
fect of FY disparity cannot be evaluated in subjects who receive
this protocol .
To the best of our knowledge, this is the first work examining the
effect of donor/recipient FY disparity on GVHD incidence after
HLA-identical sibling HSCT and our results demonstrated no
significant correlation between FY polymorphism and GVHD .
Figure 4. Univariate analysis of correlation between FY nonidentity
and incompatibility and incidence of chronic GVHD among each
HLA class I subgroup.
Figure 5. Multivariate logistic regression model of risk factors for
chronic GVHD among HLA-B7-like positive patients.
Figure 3. Univariate analysis of correlation between FY nonidentity
and incompatibility and incidence of acute GVHD among each HLA
class I subgroup. HLA, human leukocyte antigen.

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Acknowledgments
The authors thank the patients and their donors who partici-
pated in this study . The authors are also very grateful to the
research and medical staff of the National Blood Transfusion
Center and National Bone Marrow Transplantation Centre
of Tunis for their valuable technical assistance . This work was
supported by the Tunisian Ministry for Higher Education and
Scientific Research (UR06SP04) and the Tunisian Ministry of
Public Health .
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