Hum Genet (1995) 95:407-410 �9 Springer-Verlag 1995

Christophe Toumamille �9 Caroline Le Van Kim Pierre Gane �9 Jean-Pierre Cartron �9 Yves Colin

Molecular basis and PCR-DNA typing of the Fya/fyb blood group polymorphism

Received: 12 September 1994

Abs t rac t The Duffy blood group antigens are carried by the erythrocyte membrane glycoprotein gpD, which has a molecular weight of 35--45 kDa and which has been re- cently cloned. In this report, we have determined, at the nucleic acid level, the molecular basis for the blood group Fya/Fyb polymorphism. The gpD cDNAs isolated by re- verse transcription/polymerase chain reaction (RT-PCR) from Fy(a+b-) and Fy(a-b+) donors differed by only one base susbstitution (G131A) changing Gly to Asp at posi- tion 44 of the gpD protein. When expressed in simian Cos-7 cells, the Fy(a+b--) and Fy(a-b+) gpD cDNA pro- duce cell surface proteins that react with the anti-Fya and anti-Fyb antisera, respectively, demonstrating that they represent the FY*A and FY*B alleles of the Duffy blood group locus. The G131A nucleotide substitution has been correlated with a BanI restriction site polymorphism, which has allowed us to develop a method for the DNA typing of the main Duffy blood group antigens, by means of PCR/ restriction fragment length polymorphisms.

Fy(a+b+), defined with anti-Fy a and anti-Fy b anti-sera, have a frequency of 0.195, 0.33 and 0.475, respectively. The Fy allele, which produces neither Fy a nor Fy b anti- gens [Fy(a-b-) phenotype], is rare in Caucasians but rep- resents the major allele in blacks 0;requency: 0.7 to 1). The Duffy glycoprotein D (gpD), deduced from its cDNA sequence analysis (Chaudhuri et al. 1993), is a 338-amino- acid polypeptide that is probably organized into seven trans- membranous domains (Neote et al. 1994). Expression of the Fy recombinant protein in eucaryotic cells has demon- strated that the Duffy blood group antigen is the human erythrocyte chemokine receptor for a family of pro-in- flammatory peptides, including interleukin-8, melanoma growth-stimulating activity, RANTES and monocyte chemo- tactic protein-1 (MCP-1) (Chaudhuri et al. 1994; Neote et al. 1994). In this report, we describe the cloning, sequenc- ing and expression of cDNAs from donors with different Fy phenotypes in order to determine the genetic basis for the polymorphisms between FY*A and FY*B alleles of the gene encoding the Duffy blood group gpD protein.

Introduction

The Duffy blood group antigens on erythrocytes are of wide interest in clinical medicine because of their in- volvement in transfusion incompabilities and haemolytic disease of newborns (HDN) (Mollison et al. 1993) and of their association with the invasion of red cells by the Plas- modium vivax malaria parasite (Miller et al. 1976; Barn- wall et al. 1989). The Duffy locus maps to chromosome 1q22-q23 (Donahue et al. 1968) and is composed of two common alleles in Caucasians, FY*A and FY*B, coding for the Fy a and Fy b antigens, respectively, In Caucasian populations, the three phenotypes Fy(a+b-), Fy(a-b+) and

C. Toumamille �9 C Le Van Kim �9 P. Gane �9 J-P. Cartron Y. Colin (l~) Unit6 INSERM U76, Institut National de Transfusion Sanguine, Rue Alexandre Cabanel, F-75015 Paris, France

Materials and methods

Blood samples

Blood samples from healthy donors were collected on EDTA and the Fy phenotypes were determined by agglutination studies using the antiglobulin gel test (Diamed SA, Morat, Switzerland) with anti-Fy" and anti-Fy b human polyclonal antibodies (Ortho Diag- nostic Systems, Raritan, N.J., USA)

Reverse transcription coupled with polymerase chain reaction amplification and sequence analysis

Total RNAs were extracted from 400 ILl whole peripheral blood by the mini scale acid-phenol-guanidium method (Lozano et al. 1993). The reverse transcription polymerase chain reaction (RT- PCR) method was as described (Mouro et al. 1993). Primers used in the PCR were as follows: P1, 5"-TGCCCCTCAGTTCCCAGG-3" (sense, positions -40 to -23) and P2, 5 "-CACAAAGGCAGT. Thirty cycles were performed under the following conditions: 1 min at 94~ 1.5 rain at 58~ and 2 min at 72 ~ C. PCR products were subcloned

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in pUC 18 vector and sequenced on the Applied Biosystems model 373 DNA sequencer by fluorescent dye terminator cycle sequencing.

Restriction analysis of PCR-amptified genomic sequences

For the restriction fragment length polymorphism (RFLP) assay, the PCR reaction was performed with 200 ng leukocyte DNA be- tween primers P3, 5"-AACTGAGAACTCAAGTCAGC-3" (sense, positions +45 to +65) and P4, 5"-ATGAAGAAGGGCAGTGCA- GAGT3" (anti-sense, positions +203 to +184) under the following conditions; 30 cycles of 1 min at 94~ 1 min at 58~ and 1 rain at 72~ PCR products were purified on Ultrafree-MC (30000 NMWL) filter units (Millipore, Bedford, MA, USA) and one third was digested for 2 h with 20 units BanI restriction enzyme. Re- striction fragments were directly analysed in 12% acrylamide mini gels.

Construction of expression plasmid and DNA transfections

The cDNA fragments containing the entire Fy coding sequences were isolated from recombinant pUC 18 plasmid after digestion by HindllI and Asp718. Restriction fragments were blunt-ended with the T4 DNA polymerase and subcloned into the EcoRV-digested pcDNA-1 expression vector (Invitrogen). Cos-7 cells grown in Iscove medium supplemented with 10% fetal calf serum were transfected by electroporation (at 200 V, 960 gF) in 180 ~1 phos- phate-buffered saline (PBS) with HEPES (10 mM, pH 7.4) using 10 7 cells/assay with 10 ~tg recombinant plasmid. Transfectants were analysed after 36 h.

Flow cytometry analysis

The expression of Duffy antigens was tested by staining the cells with anti-Fy a and anti-Fy b human antisera (Ortho Diagnostic Sys- tems, Raritan, N.J., USA) and with a murine monoclonal anti-F6 antibody, mAb i3A (Riwom et al. 1994). Cells (3 • 105) were in- cubated for 60 min at 22~ with antibodies diluted 1/2 in 0.15 M PBS. After being washed with PBS supplemented with 0.5% bovine serum albumin, the cell suspension was incubated with flu- orescein-conjugated anti-mouse or anti-human lgG (H+L) (Im- munotech, Marseille, France). After another washing step, 0.1 ng propidium iodide (Pl) was added to 1 ml cell suspension. PI-posi- tive cells (dead cells) were excluded from analysis. Fluorescence was measured on a FACScan flow cytometer (Becton Dickinson, San Jose, Calif., USA).

Results and discussion

Ampl i f i ca t ion and sequencing of al lel ic Fy c D N A s

R N A s extracted from whole b lood of individuals with the F y ( a + b - ) , F y ( a - b + ) , Fy(a+b+) and F y ( a - b - ) phenotypes were used as templates to ampl i fy the entire Fy coding re- gion be tween pr imers P1 and P2 (Fig. 1) specific for the 5" and 3" non-coding regions of the Fyb71-81 c D N A clones obta ined from human bone marrow m R N A s of F y ( a - b + ) individuals (Chaudhuri et al. 1993). In all samples, a PCR product with the expected size of 1105 bp was obtained (data not shown). The sequence of the Fy c D N A s der ived from two unrelated F y ( a - b + ) donors was identical to that of the Fyb71-81 clones. Sequence analysis of the Fy cD- NAs obtained from two F y ( a + b - ) samples revealed only one substitution at nucleotide 131 (position +1 being taken as the first nucleot ide in the init iator codon) when com- pared with the 1014 nucleot ides of the coding region of the Fy (a -b+) cDNAs. Nucleot ides at posit ion 131 in the cDNAs of Fy (a+b- ) and F y ( a - b + ) were G and A, respec- tively, cDNAs clones der ived from Fy(a+b+) m R N A s ex- hibi ted ei ther a G or an a residue at posi t ion 131. These resul ts s t rongly sugges ted that the FY*A and FY*B al le- les differ by the single G131A substi tution, which results in a Gly to Asp amino acid change at posi t ion 44 of the Duffy gpD protein. Interest ingly, this posi t ion lies within the 66-residue NH2 terminal region of the gpD protein, a region that has been predic ted to be on the exoce l lu la r side of the cell membrane (Chaudhuri et al. 1993; Neote et al. 1994), and that is therefore avai lable , on intact red cells to ant i -Fy antibodies.

Al though the Fy transcripts from F y ( a - b - ) samples were undetectable in Northern blot analysis (Chaudhuri et al. 1993), they have been successful ly PCR-ampl i f i ed in the course of this s tudy (not shown). Nucleot ide se- quences obtained from one F y ( a - b - ) re t iculocyte sample have revealed no mutat ions compared with the F y ( a - b + ) sequence. This result supports previous f indings indicat-

Fig. l Schematic representa- tion of the gpD cDNAs iso- lated by RT-PCR from whole blood of Fy-typed donors. Solid bar and thin lines re- present the coding and non- coding sequences, respectively. Positions of primers PI and P2 used in PCR are indicated. The nucleotide sequence of Fy(a+b-) and Fy(a-b+) clones were identical, except for the GI31 A substitution (*) result- ing in the Gly44Asp polymor- phism on the gpD protein. Se- quencing of several clones de- rived from Fy(a+b+) samples revealed either a G or a A residue at position 134

P1

Fy (a+ b-)

Fy (a- b+)

gpD transcripts

+1 131 +1014

* l li:i~ i ~il ~i~i~!ii~i~;~ili~:.;:.ii~i~i~ i i ~ ~il ~:.~ ~::i~ !!!!i! !i! !!!! !i!ii ~ii~ii:.iiii~ii!i:.~ :.i:.~ii~i~ii::~ii:,ii;:.~i~:.~!~ ~ ~'~!~ ~ !~ ! ~.!~:~ :.~i:.;~:.~i 4 - - , -

P2 1105 bp =

Fy (a+ b+)

anti-Fya anti-Fyb I i i. i

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2% F

0 4 ] 0 4

Log Fluorescence

A

1) PCR P3

,~ 2) Banl RFLP "11

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I

B Z > I

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O O Z > , . I ,

Fig. 2 Expression of the Fy a and Fy b blood group antigens in Cos- 7 cells. Cos-7 cells were transfected with plasmid containing either the FY*A (pcDNA-Fya) or the FY*B (pcDNA-Fyb) allelic cD- NAs of the Duffy gpD protein, or with the pcDNA1 vector alone as a negative control. Expression of the Fy a and Fy b antigens was determined by flow cytometry analysis, using anti-Fy a and anti-Fy b human antisera. The relative numbers of positive cells in the se- lected windows are indicated as percentages

409

I gpD gene

159 bp P4

86 bp I 73 bp I

159 bp

FY*A Allele

FY*B Allele

Fig.3 A, B Fya/Fyb DNA typing. A Strategy of the PCR-RFLP. Primers P3 and P4 were designed to amplify a 159-bp gpD gene fragment that encompassed the single base substitution (*) identi- fied between the FY*A and FY*B alleles and that was correlated with an allele-specific BanI restriction site. B The DNA from donors with the indicated Duffy phenotypes (lanes 1-10) was used as templates in the PCR-RFLP assay. Restriction fragments were separated on 12% acrylamide gel

ing that Duffy-negative individuals possess a normal structural gpD gene but that they do not express gpD pro- tein because they do not efficiently synthesize Duffy-spe- cific mRNA (Chaudhuri et al. 1993).

Expression ot the Fya and Fyb antigens in transfected Cos-7 cells

It has been previously shown that recombinant gpD pro- tein can be expressed at the cell membrane of human K562 and 293 cell lines (Chaudhuri et al. 1994; Neote et al. 1994); it can be detected by the murine monoclonal an- tibody, anti-Fy6, which defines a non-polymorphic anti- genic determinant that is present in all Duffy-positive cells but that is absent in Fy(a-b-) cells (Nichols et al. 1987). We have now transfected simian Cos-7 cells with expression vectors containing the Fya (pcDNA-Fya) or Fyb (pcDNA-Fyb) allelic cDNAs, as defined above. Flow cytometry analysis revealed positive staining with the i3A

anti-Fy6 antibody on cells transfected either with pcDNA- Fya or pcDNA-Fyb, indicating that, as previously re- ported for others host cells, the recombinant gpD protein is readily transported into the membrane of Cos-7 trans- fected cells (not shown). In contrast, when anti-Fy a or anti-Fy b antisera were used, positive signals were ob- tained only with cells transfected with pcDNA-Fya or pcDNA-Fyb, respectively (Fig. 2). As. a negative control, no staining was observed on cells transfected with the pcDNA1 vector alone or when a non-immune serum was used instead of the anti-Fy antibodies. These results indi- cate that the Gly44Asp substitution in the gpD protein is responsible for the Fya/Fyb antigenic polymorphism of the Duffy blood group system.

Fya and Fyb DNA typing by PCR-RFLP

The substitution identified at nucleotide 131 of the Fy mRNAs was correlated with the presence or the absence

410

of a BanI restriction site (GGTGCC--~GATGCC) on the FY*A and FY*B alleles defined above. This observation was used to develop a PCR-RFLP assay for the DNA typ- ing of the common Fy phenotypes. Primers P3 and P4 were designed to amplify a 159-bp fragment encompass- ing this polymorphic posit ion on the Fy cDNA. The same PCR product was obtained with genomic DNA as the tem- plate, indicating that the Fy coding sequence is not dis- rupted by intervening sequences in this region of the gpD gene. PCR-RFLP of genomic DNAs from 10 unrelated donors of each common Fy phenotypes has been carried out and typical results are shown in Fig. 3. After BanI di- gestion, the 159-bp PCR product was cleaved into two fragments of 86 and 79 bp in all Fy(a+b- ) samples. Only the uncleaved 159-bp fragment was observed with the DNAs from Fy(a -b+) donors. The 159-bp, 86-bp and 79- bp fragments were all detected in the heterozygous Fy(a+b+) samples.

In conclusion, we have demonstrated, by nucleotide sequencing and eukaryotic expression, that the pheno- types given by FY*A and FY*B alleles are produced by the Gly44 Asp amino acid substitution resulting from the nucleotide polymorphism G 131A. The BanI RFLP caused by this difference makes it possible to determine the Fy phenotype by DNA typing when intact red blood cells are not available for classical serological studies. Such PCR- based methods have been recently reported for the detec- tion of RHD, RHc and RHE alleles in fetal cells from am- niotic fluid of Rh- immunized pregnant woman (Bennet et al. 1993; Le Van Kim et al. 1994). Since anti-Fy antibod- ies can also cause HDN (Mollison et al. 1993), it is ex- pected that the ability to determine the Fya/Fy b status of fetuses from Fy- immunized mothers will prove useful in the management of pregnancies at risk of Fy haemolytic disease.

Acknowledgements We thank Dr. Van Huffel (Paris) for the gift of genomic DNAs from Fy-typed donors and Prof. Galacteros (Creteil) for providing Fy(a-b-) blood samples. We are grateful to Dr. Blanchard (Nantes) for the gift of the i3A anti-Fy6 monoclonal antibody. This work was supported in part by the Institut National de la Sant6 et de la Recherche M6dicale (INSERM) and the Caisse Nationale d'Assurances Maladies des Travailleurs Salari6s (CNAMTS).

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