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The Duffy blood group system in the Tunisian population Le groupe sanguin Duffy dans la population tunisienne M. Ouchari , H. Romdhane , T. Chakroun , S. Abdelkefi , I. Jarrey , B. Houissa , S. Jemni Yacoub ∗ Centre régional de transfusion sanguine, hôpital Farhat Hached, Sousse, Tunisia
Abstract Background. – Tunisia was described to as genetically heterogenous. Besides the 1% native Berber, the genetically influence of the Europeans seems much larger than that of sub-Saharan populations. Due to their ethnic variability, blood group variants have the potential to support population analyses. The aim of this study was to estimate the Duffy blood group system in this mixed population with enhanced characterization of samples with aberrant expression. Materials and methods. – Standard serological testing for the Duffy antigen was done for 105 Tunisian blood donors. Samples with altered Fy expression underwent DNA sequencing of the DARC, RHD and RHCE genes. Results. – The Fy(a-b+) was the most common phenotype identified in the Tunisian population (38.1%). Five samples with Fy(a-b-) phenotype were determined as FY*02N.01/FY*02N.01 by a homozygous occurrence of the FY*B-67C>T alteration. Another three individuals exhibited a Fy(b+w )Fyx expression, confirmed by a FY*A/FY*02M.01 (n = 1) and a FY*02M.01/FY*02M.01 (n = 2) genotype. RHD and RHCE sequencing (n = 8) revealed altered alleles observed in black populations in 5 samples. One individual with FY*02M.01/FY*02M.01 have the silent 165C>T nucleotide substitution each in the RHD and RHCE gene. Discussion. – The composition of blood group variants determined in this study confirms the genetically proximity of Tunisia to Europe. The small sub-Saharan genetic influence was approved by a limited number of variant samples associated with the black population. © 2015 Elsevier Masson SAS. All rights reserved. Keywords: Duffy; Aberrant RH alleles; Tunisia
Résumé Contexte. – La Tunisie est considérée comme génétiquement hétérogène. À côté d’une population Berbère initiale évaluée à 1 %, l’influence génétique issue des Européens semble supérieure à celle issue des populations sub-sahariennes. Du fait de leur variabilité ethnique, les groupes sanguins sont utiles dans les analyses de population. Cette étude a pour but de caractériser le système de groupe sanguin Duffy dans cette population métissée, en s’attachant aux cas des allèles aberrants. Matériel et méthodes. – Cent cinq donneurs de sang tunisiens pris au hasard ont été testés en sérologie classique pour le groupe sanguin Duffy. Les cas présentant une expression altérée ont subi un séquenc¸age ADN des gènes DARC, RHD et RHCE. Résultats. – Le phénotype Fy(a-b+) est le plus fréquent dans la population tunisienne (38,1 %). Cinq échantillons Fy(a-b-) ont été trouvés FY*02N.01/FY*02N.01 du fait d’une altération homozygote FY*B-67C>T. Trois autres cas avec une expression Fyb+w/Fyx avaient un génotype FY*A/FY*02M.01 (1 cas) et FY*02M.01/FY*02M.01 (2 cas). Le séquenc¸age RHD et RHCE fait sur 8 échantillons a montré des allèles altérés analogues à ceux des populations négroïdes dans 5 cas. Une substitution muette 165C>T a été trouvée dans les deux gènes RHD et RHCE d’un individu FY*02M.01/FY*02M.01. Discussion. – Les variants de groupes sanguins retrouvés dans cette étude confirment la proximité génétique de la Tunisie avec l’Europe. Une influence sub-saharienne plus faible est retrouvée avec certains variants associés à la population négroïde. © 2015 Elsevier Masson SAS. Tous droits réservés. Mots clés : Duffy ; Allèle RH aberrant ; Tunisie ∗
Corresponding author. E-mail address: [email protected] (S. Jemni Yacoub).
http://dx.doi.org/10.1016/j.tracli.2015.03.002 1246-7820/© 2015 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Ouchari M, et al. The Duffy blood group system in the Tunisian population. Transfusion Clinique et Biologique (2015), http://dx.doi.org/10.1016/j.tracli.2015.03.002
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1. Introduction Tunisia, located in North Africa, is believed to be inhabited by Berbers since from at least 10,000 B.C. Seafaring peoples like the Phoenicians, Vandals, Greeks, French and Romans facilitated the migration across the Mediterranean. Lastly, the Islamic influence of the “Moors” from Southern Spain and several Arab occupations have shaped Tunisia. Genetically, about 1% of the global Tunisian population are native Berber [1–3]. X-chromosomal based analysis demonstrated a smaller subSaharan genetic influence and elucidated a relative genetically heterogeneity within current Tunisian populations [4]. Due to their ethnic variability, blood group variants may support human variation studies. The Duffy (FY) blood group system encodes the DARC glycoprotein, which acts as an erythroid receptor for the malaria parasites Plasmodium vivax and P. knowlesi [5] and a chemokine receptor of both C-X-C and C-C classes [6]. Two major antigens, Fya and Fyb , are encoded by the FY*A syn. FY*01 and FY*B syn. FY*02 allele, differing by a single amino acid change in the DARC exon 2 [7,8]. While the Fya and Fyb phenotypes are distributed equally among Caucasians, Blacks predominantly express the Fyb antigen [9]. Homozygosity or compound heterozygosity of the FY*02N.01 allele results in a Fy(a-b-) phenotype frequently observed in the black population. Its molecular basis is a -67 T>C substitution in the GATA-1 binding motif of the DARC promoter causing gene-silencing [8]. First discovered in persons of African descent associated with the FY*B allele and therefore called FY*B ES , and subsequently the same mutation was later determined to be linked to the FY*A haplotype in individuals living in a Plasmodium vivaxendemic region of Papua New Guinea and therefore annotated FY*A ES [10]. In addition, rare cases of Fy(a-b-) among the Caucasian population were described with a different molecular background [11]. Carriers with diminished expression of the Fyb antigen annotated Fy b+w Fyx were shown to harbour an altered FY*B genotype with the specific Arg89Cys and Ala100Thr amino acid substitutions [12,13]. An additional alteration, Ala49Ser, was described for some individuals with the FyX phenotype, annotated FY*02M.02 [12,14]. The present study attempts to elucidate the Duffy phenotype composition among a Tunisian multiethnic donor cohort and to further characterize those samples with aberrant Fy expression with regard to the RH blood group system. 2. Materials and methods 2.1. Blood samples EDTA blood samples of 105 random blood donors from the Sahel were collected at the Regional Blood Transfusion Center of Sousse, Tunisia. 2.2. Serologic testing Duffy phenotyping of fresh blood samples was done by agglutination tests with anti-Fy(a)FY1 IgG (lot FYA226AX)
and anti-Fy(b)FY2 IgG (lot FYB075BX) human antibodies (Ortho Clinical Diagnostics, Neckargemund, Germany) using ID coombs cards (anti-IgG/C3d) (Bio-Rad Laboratories GmbH, Vienna, Austria). 2.3. Molecular analysis Genomic DNA of 9 samples exhibiting an altered Fy phenotype was automatically isolated from 200 L of EDTA blood using the MagNA Pure Compact DNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. For amplification of a DARC gene segment encompassing exons 1 and 2, primers 5 -GTTCAAGGGGATGGAGGAGC-3 and 5 TCATCATTCACCTTCTTCCCAA-3 [15] were introduced. The PCR was carried out in a LightCycler 2.0 instrument (Roche Diagnostics GmbH, Manheim, Germany) and consisted of an initial denaturation of 2 minutes at 95 ◦ C, followed by 35 cycles of 30 seconds at 95 ◦ C, 50 seconds at 60 ◦ C, 2 minutes at 72 ◦ C and a final elongation of 7 minutes at 72 ◦ C. A 25 L PCR mixture contained 1 × High Fidelity Enyzme buffer, 0.25 mol/L each primer, 0.2 mM dNTPs, 1U High Fidelity Expand enzyme (Roche Diagnostics GmbH, Manheim, Germany) and 10 ng of DNA. RHD and RHCE exons 1 to 10 including intron/exon boundaries were amplified as published before [16,17]. PCR products were treated with alkaline phosphatase and exonuclease I (ExoSAP-IT, High Wycombe, UK) before analyzing by an automated ABI 3130 sequencer (Life Technologies, Vienna, Austria) using BigDye Chemistry (BigDye Terminator v1.1 Cycle Sequencing Kit, Life Technologies). Data analysis was performed with SeqScape v. 2.7 (Life Technologies). 3. Results In this study, we verified six Fy phenotypes among 105 samples, comprising Fy(a-b+) (n = 40, 38.1%), Fy(a+b-) (n = 24, 22.9%), Fy(a+b+) (n = 32, 30.5%), Fy(a-b-) (n = 5, 4.8%), Fy(ab+w ) Fyx (n = 3, 2.9%), Fy(a+b+w ) Fyx (n = 1, 1%). Standard serological Fy typing revealed a diminished expression of the Fyb antigen in 4 samples and a Fy(a-b-) phenotype in 5 individuals (Table 1). Those samples underwent molecular characterization by nucleotide sequencing of the DARC gene. The FY*02M.01 gene formation was determined by DARC(Arg89Cys,Ala100Thr) in a homozygous (n = 2) and heterozygous occurrence (n = 1) (Table 2). In one sample with diminished Fyb phenotype, no alteration of the DARC gene Table 1 Results of serological Duffy typing. Duffy phenotype
n
%
Fy(a+b+) Fy(a+b-) Fy(a-b+) Fy(a-b-) Fy(a-b+w )Fyx Fy(a+b+w )Fyx
32 24 40 5 3 1
30.5 22.9 38.1 4.8 2.9 1.0
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Table 2 Molecular DARC, RHD and RHCE typing results of samples with aberrant Fy phenotype. Fy phenotype
FY genotype
RHD genotype
RHCE genotype
Fy(a-b-)
FY*02N.01/FY*02N.01 FY*02N.01/FY*02N.01 FY*02N.01/FY*02N.01 FY*02N.01/FY*02N.01 FY*02N.01/FY*02N.01 FY*A/FY*02M.01 FY*02M.01/FY*02M.01 FY*02M.01/FY*02M.01
RHD/RHD*01N.01 RHD*01N.01/RHD*01N.01 RHD/RHD*01N.01 RHD/RHD*01N.01 RHD/RHD*01N.01 RHD/RHD*01N.01 RHD*DAU-3/RHD*01N.01 RHD(165C>T)/RHD
RHCE*Ce/ceVS.02 RHCE*ce/ce01.06 RHCE*Ce/ce01.06 RHCE*Ce/Ce RHCE*Ce/ce RHCE*ce/ceVS.01 RHCE*Ce/ce RHCE*cE/Ce(165C>T, 486 C>T)
Fy(a+b+w ) Fyx Fy(a-b+w ) Fyx Fy(a-b+w ) Fyx
was determined. Further serological characterization revealed a positive DAT, accounting for the inconclusive Fy typing results. All 5 samples without expression of the Fy antigen were shown to harbour the 67C>T mutation in the GATA-1 binding motif of the FY*B promoter in a homozygous occurrence associated with a FYnull phenotype (Table 2). Molecular characterization of the RH genes revealed a RHD gene deletion in one sample associated with a RHCE*ce/ce01.06 allele encoded by RHCE(Ala85Gly). The other 4 individuals carried a common hemizygous RHD allele. The presence of RHCE*Ce/ceVS.02 was determined in one sample by RHCE(Trp16Cys,Leu245Val) (Table 2). One individual with a Fy(a+b+w ) phenotype exhibited a RHCE*ceVS.01 (Leu245Val) with RHCE*ce on the partner chromosome. No alterations were determined in its RHD gene sequence. Out of two individuals with Fy(a-b+w ), one was shown to exhibit a RHD*DAU-3 characterized by RHD(Val279Met, Thr379Met) and a RHCE*Ce/ce. The second sample harbored a heterozygous nucleotide alteration 165C>T in RHD exon 2. Interestingly, the RHCE allele of this sample also revealed the 165C>T mutation in a heterozygous occurrence. In addition, another silent nucleotide substitution was located in exon 3, RHCE(486C>T). No alteration in the RhCE expression was observed, probably due to a normal RHCE in trans. 4. Discussion The most common phenotype Fy(a-b+) occured with a frequency of 38.1% similar to that reported for Caucasians (34%), but much higher than that of 22% described for the Black population [9]. Among 105 Tunisian blood donor samples, 5 (4.8%) harbored a FYnull phenotype, caused by a homozygous FY*02N.01 genotype. This allele is uncommon in the Caucasian population, whereas it was described with a frequency of 68% in Blacks [9] and in 11.3% in a previous study of Tunisians blood donors [18]. The African sub-saharan genetic contribution in our cohort seems low than in the previous study [18]. This disparity may be explained by the different sampling area and the sampling size of our study. We not confirm the FY*01.N.01 previously described in our population [18], but the FY null phenotype described in this presented work is due to FY*02N.01 genotype which is frequently observed in the black population [8]. Standard serological Fy typing showed the presence of 4 samples with diminished expression of Fyb antigen Nucleotide
sequencing of these samples confirmed the FY*02M.01 allele in 3 individuals. Samples with a diminished expression or lack of the Fy antigen underwent DNA sequencing of RHD and RHCE exons 1-10. Not surprisingly, aberrant gene formations associated with Black populations were confirmed among those samples. The RHCE*ceVS.02 allele is encoded by two mutations, 48G>C and 733C>G, and leads to expression of V, VS and hrB [19]. In the black population, the 48G>C substitution is common in a ce haplotype [20]. The 733C>G substitution in RHCE exon 5 leads to the Leu245Val amino acid change and forces the expression of the VS antigen [19–21] which is extremely rare in people of European and Asian origin but has a frequency of up to 50% in African descents [19–21]. We suggest that for the Tunisian region, pre-transfusion testing should include an antibody screening test for the VS antigen. The RHCE*ce01.06 allele results from a single point mutation in RHCE exon 2 (254C>G) leading to a Ala85Gly substitution in the third transmembrane domain. It encodes partial e and showes the absence of the CEAG and hrB antigens [9]. In our case, the presence of a normal RHCE*ce in trans masked an aberrant RhCE expression. One sample with Fy(a-b+w ) was associated with RHD(165C>T)/RHD and RHCE*cE/Ce(165C>T, 486C>T). The RHD(165C>T) allele was annotated before to occur with a normal D in an Austrian patient (accession no. FN545815), a weakened D in a patient from China (accession no. DQ309583) and in trans to a normal D in one Tibetian individual (AM398143). Interestingly, the RHCE allele of this sample also revealed the 165C>T mutation in a heterozygous occurrence. In our study, this sample expressed a normal D phenotype. A possible weakened D antigen would have been masked by the normal RHD in trans. To our knowledge, the associated RHCE(165C>T, 486C>T) allele was not determined before. Both nucleotide alterations do not lead to an amino acid substitution, and we could not find any influence on the RhCE. Several studies have been undertaken to describe the genetic structure of Tunisian populations employing genetic markers specific for immunoglobulin chains [22], HLA class II [23], autosomal short tandem repeats (STRs) [24], and polymorphic Alu insertions [25]. Our findings underline the results of genetically based population studies which indicate that the Tunisian genetic distances to Europa are small [26]. This may be easily explained by the eventful history of the country concerning occupations and immigrations. The small sub-Saharan genetic
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influence in Tunisia [26] was confirmed by a limited number of blood group variants associated with the black population. Funding The authors declare no competing financial interests. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments We are grateful to all blood donors who contributed their blood samples. We thank Helene Polin, Christian Gabriel, Martin Danzer, Melanie Brisner, Anna Reiter, Waltraud Gaszner for their help. This work was supported by UR06SP05 Centre Régional de Transfusion Sanguine de Sousse and Red Cross Transfusion Service of Upper Austria. References [1] Sanchez-Mazas A. The Berbers of North Africa: genetic relationships according to HLA and other polymorphisms. In: Arnaiz-Villena A, editor. Prehistoric Iberia: genetics, anthropology and linguistic. New York: Kluwer Academic; 2000. p. 65–77. [2] Luis JR, Rowold DJ, Regueiro M, Caeiro B, Cinnioglu C, Roseman C, et al. The Levant versus the Horn of Africa: evidence for bidirectional corridors of human migrations. Am J Hum Genet 2004;74:532–44. [3] Fadhlaoui-Zid K, Buhler S, Dridi A, El Gaaied AB, Sanchez-Mazas A. Polymorphism of HLA class II genes in Berbers from Southern Tunisia. Tissue Antigens 2010;76:416–20. [4] Tomas C, Sanchez JJ, Barbaro A, Brandt-Casadevall C, Hernandez A, Ben Dhiab M, et al. X-chromosome SNP analyses in 11 human Mediterranean populations show a high overall genetic homogeneity except in North-west Africans (Moroccans). BMC Evol Biol 2008;8:75. [5] Miller LH, Mason SJ, Dvorak JA, McGinniss MH, Rothman IK. Erythrocyte receptors for (Plasmodium knowlesi) malaria: Duffy blood group determinants. Science 1975;189:561–3. [6] Hadley TJ, Peiper SC. From malaria to chemokine receptor: the emerging physiologic role of the Duffy blood group antigen. Blood 1997;89:3077–91. [7] Iwamoto S, Omi T, Kajii E, Ikemoto S. Genomic organization of the glycoprotein D gene: Duffy blood group Fya/Fyb alloantigen system is associated with a polymorphism at the 44-amino acid residue. Blood 1995;85: 622–6. [8] Tournamille C, Colin Y, Carton JP, Le Van Kim C. Disruption of a GATA motif in the Duffy gene promoter abolishes erythroid gene expression in Duffy negative individuals. Nat Genet 1995;10:224–8. [9] Reid ME, Lomas-Francis C, Olsson ML. The blood group antigen facts book. 3rd ed; 2012, ISBN 978-0-12-415849-8.
[10] Zimmerman PA, Wooley I, Masinde GL, Miller SM, McNamara DT, Hazlett F, et al. Emergence of FY* Anull in a Plasmodium vivax endemic region of Papua New Guinea. Proc Natl Acad Sci U S A 1999;96:13973–7. [11] Rios M, Chaudhuri A, Mallinson G, Sausais L, Gomensoro-Garcia AE, Hannon J, et al. New genotypes in Fy(a-b-) individuals: nonsense mutation (Trp to stop) in the coding sequence of either FYA or FYB. Br J Haematol 2000;108:448–54. [12] Olsson ML, Smythe JS, Hansson C, Poole J, Mallinson G, Jones J, et al. The Fy(x) phenotype is associated with a missense mutation in the Fy(b) allele predicting Arg89Cys in the Duffy glycoprotein. Br J Haematol 1998;103:1184–91. [13] Tournamille C, Le Van Kim C, Gane P, Le Pennec PY, Roubinet F, Babinet J, et al. Arg89Cys substitution results in very low membrane expression of the Duffy antigen/receptor for chemokines in Fy(x) individuals. Blood 1998;92:2147–56. [14] Parasol N, Reid M, Rios M, Castilho L, Harari I, Kososwer N. A novel mutation in the coding sequence of the FY*B allele of the Duffy chemokine receptor gene is associated with an altered erythrocyte phenotype. Blood 1998;92:2237–43. [15] Gassner C, Kraus RL, Dovc T, Kilga-Nogler S, Utz I, Mueller TH, et al. Fyx is associated with two missense point mutations in its 17 gene and can be detected by PCR-SSP. Immunohematology 2000;16:61–7. [16] Strobel E, Noizat-Pirenne F, Hofmann S, Cartron JP, Bauer MF. The molecular basis of the Rhesus antigen Ew. Transfusion 2004;44:407–9. [17] Polin H, Danzer M, Hofer K, Gassner W, Gabriel C. Effective molecular RHD typing strategy for blood donations. Transfusion 2007;47:1350–5. [18] Sellami MH, Kaabi H, Midouni B, Dridi A, Mojaat N, Boukef MK, et al. Duffy blood group system genotyping in an urban Tunisian population. Ann Hum Biol 2008;35:406–15. [19] Pham BN, Peyrard T, Juszczak G, Dubeaux I, Gien D, Blancher A, et al. Heterogeneous molecular background of the weak C, VS+, hrB-, HrB phenotype in black persons. Transfusion 2009;49:495–504. [20] Noizat-Pirenne F, Lee K, Le Pennec PY, Simon P, Kazup P, Bachir D, et al. Rare RHCE phenotypes in black individuals of Afro-Caribbean origin: identification and transfusion safety. Blood 2002;100:4223–31. [21] Daniels GL, Faas BH, Green CA, Smart E, Maaskant-van Wijk PA, Avent ND, et al. The VS and V blood group polymorphisms in Africans: a serologic and molecular analysis. Transfusion 1998;38:951–8. [22] Loueslati BY, Sanchez-Mazas A, Ennafaa H, Marrakchi R, Dugoujon JM, Lefranc JM, et al. A study of Gm allotypes and immunoglobulin heavy gamma IGHG genes in Berbers. Arabs and sub-Saharan Africans from Jerba Island, Tunisia. Eur J Immunogenet 2001;28:531–8. [23] Guenounou BA, Loueslati BY, Buhler S, Hmida S, Ennafaa H, Khodjetel-Khil H, et al. HLA class II genetic diversity in southern Tunisia and the Mediterranean area. Int J Immunogenet 2006;33:93–103. [24] Bosch E, Clarimon J, Perez-Lezaun A, Calafell F. STR data for 21 loci in northwestern Africa. Forensic Sci Int 2001;116:41–51. [25] Ennafaa H, Ben Amor M, Yacoubi-Loueslati B, Khodjet-el-Khil H, Gonzalez-Perez E, Mora P, et al. Alu polymorphisms in Jerba Island population (Tunisia): comparative study in Arab and Berber groups. Ann Hum Biol 2006;33:634–40. [26] Athanasiadis G, Esteban E, Vi M, Dugoujon J-M, Moschona N, Chaabani H, et al. The X chromosome Alu insertions as a tool for human population genetics: data from European and African human groups. Eur J Hum Genet 2007;15:578–83.
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The Duffy blood group system in the Tunisian population Le groupe sanguin Duffy dans la population tunisienne M. Ouchari , H. Romdhane , T. Chakroun , S. Abdelkefi , I. Jarrey , B. Houissa , S. Jemni Yacoub ∗ Centre régional de transfusion sanguine, hôpital Farhat Hached, Sousse, Tunisia
Abstract Background. – Tunisia was described to as genetically heterogenous. Besides the 1% native Berber, the genetically influence of the Europeans seems much larger than that of sub-Saharan populations. Due to their ethnic variability, blood group variants have the potential to support population analyses. The aim of this study was to estimate the Duffy blood group system in this mixed population with enhanced characterization of samples with aberrant expression. Materials and methods. – Standard serological testing for the Duffy antigen was done for 105 Tunisian blood donors. Samples with altered Fy expression underwent DNA sequencing of the DARC, RHD and RHCE genes. Results. – The Fy(a-b+) was the most common phenotype identified in the Tunisian population (38.1%). Five samples with Fy(a-b-) phenotype were determined as FY*02N.01/FY*02N.01 by a homozygous occurrence of the FY*B-67C>T alteration. Another three individuals exhibited a Fy(b+w )Fyx expression, confirmed by a FY*A/FY*02M.01 (n = 1) and a FY*02M.01/FY*02M.01 (n = 2) genotype. RHD and RHCE sequencing (n = 8) revealed altered alleles observed in black populations in 5 samples. One individual with FY*02M.01/FY*02M.01 have the silent 165C>T nucleotide substitution each in the RHD and RHCE gene. Discussion. – The composition of blood group variants determined in this study confirms the genetically proximity of Tunisia to Europe. The small sub-Saharan genetic influence was approved by a limited number of variant samples associated with the black population. © 2015 Elsevier Masson SAS. All rights reserved. Keywords: Duffy; Aberrant RH alleles; Tunisia
Résumé Contexte. – La Tunisie est considérée comme génétiquement hétérogène. À côté d’une population Berbère initiale évaluée à 1 %, l’influence génétique issue des Européens semble supérieure à celle issue des populations sub-sahariennes. Du fait de leur variabilité ethnique, les groupes sanguins sont utiles dans les analyses de population. Cette étude a pour but de caractériser le système de groupe sanguin Duffy dans cette population métissée, en s’attachant aux cas des allèles aberrants. Matériel et méthodes. – Cent cinq donneurs de sang tunisiens pris au hasard ont été testés en sérologie classique pour le groupe sanguin Duffy. Les cas présentant une expression altérée ont subi un séquenc¸age ADN des gènes DARC, RHD et RHCE. Résultats. – Le phénotype Fy(a-b+) est le plus fréquent dans la population tunisienne (38,1 %). Cinq échantillons Fy(a-b-) ont été trouvés FY*02N.01/FY*02N.01 du fait d’une altération homozygote FY*B-67C>T. Trois autres cas avec une expression Fyb+w/Fyx avaient un génotype FY*A/FY*02M.01 (1 cas) et FY*02M.01/FY*02M.01 (2 cas). Le séquenc¸age RHD et RHCE fait sur 8 échantillons a montré des allèles altérés analogues à ceux des populations négroïdes dans 5 cas. Une substitution muette 165C>T a été trouvée dans les deux gènes RHD et RHCE d’un individu FY*02M.01/FY*02M.01. Discussion. – Les variants de groupes sanguins retrouvés dans cette étude confirment la proximité génétique de la Tunisie avec l’Europe. Une influence sub-saharienne plus faible est retrouvée avec certains variants associés à la population négroïde. © 2015 Elsevier Masson SAS. Tous droits réservés. Mots clés : Duffy ; Allèle RH aberrant ; Tunisie ∗
Corresponding author. E-mail address: [email protected] (S. Jemni Yacoub).
http://dx.doi.org/10.1016/j.tracli.2015.03.002 1246-7820/© 2015 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Ouchari M, et al. The Duffy blood group system in the Tunisian population. Transfusion Clinique et Biologique (2015), http://dx.doi.org/10.1016/j.tracli.2015.03.002
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1. Introduction Tunisia, located in North Africa, is believed to be inhabited by Berbers since from at least 10,000 B.C. Seafaring peoples like the Phoenicians, Vandals, Greeks, French and Romans facilitated the migration across the Mediterranean. Lastly, the Islamic influence of the “Moors” from Southern Spain and several Arab occupations have shaped Tunisia. Genetically, about 1% of the global Tunisian population are native Berber [1–3]. X-chromosomal based analysis demonstrated a smaller subSaharan genetic influence and elucidated a relative genetically heterogeneity within current Tunisian populations [4]. Due to their ethnic variability, blood group variants may support human variation studies. The Duffy (FY) blood group system encodes the DARC glycoprotein, which acts as an erythroid receptor for the malaria parasites Plasmodium vivax and P. knowlesi [5] and a chemokine receptor of both C-X-C and C-C classes [6]. Two major antigens, Fya and Fyb , are encoded by the FY*A syn. FY*01 and FY*B syn. FY*02 allele, differing by a single amino acid change in the DARC exon 2 [7,8]. While the Fya and Fyb phenotypes are distributed equally among Caucasians, Blacks predominantly express the Fyb antigen [9]. Homozygosity or compound heterozygosity of the FY*02N.01 allele results in a Fy(a-b-) phenotype frequently observed in the black population. Its molecular basis is a -67 T>C substitution in the GATA-1 binding motif of the DARC promoter causing gene-silencing [8]. First discovered in persons of African descent associated with the FY*B allele and therefore called FY*B ES , and subsequently the same mutation was later determined to be linked to the FY*A haplotype in individuals living in a Plasmodium vivaxendemic region of Papua New Guinea and therefore annotated FY*A ES [10]. In addition, rare cases of Fy(a-b-) among the Caucasian population were described with a different molecular background [11]. Carriers with diminished expression of the Fyb antigen annotated Fy b+w Fyx were shown to harbour an altered FY*B genotype with the specific Arg89Cys and Ala100Thr amino acid substitutions [12,13]. An additional alteration, Ala49Ser, was described for some individuals with the FyX phenotype, annotated FY*02M.02 [12,14]. The present study attempts to elucidate the Duffy phenotype composition among a Tunisian multiethnic donor cohort and to further characterize those samples with aberrant Fy expression with regard to the RH blood group system. 2. Materials and methods 2.1. Blood samples EDTA blood samples of 105 random blood donors from the Sahel were collected at the Regional Blood Transfusion Center of Sousse, Tunisia. 2.2. Serologic testing Duffy phenotyping of fresh blood samples was done by agglutination tests with anti-Fy(a)FY1 IgG (lot FYA226AX)
and anti-Fy(b)FY2 IgG (lot FYB075BX) human antibodies (Ortho Clinical Diagnostics, Neckargemund, Germany) using ID coombs cards (anti-IgG/C3d) (Bio-Rad Laboratories GmbH, Vienna, Austria). 2.3. Molecular analysis Genomic DNA of 9 samples exhibiting an altered Fy phenotype was automatically isolated from 200 L of EDTA blood using the MagNA Pure Compact DNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. For amplification of a DARC gene segment encompassing exons 1 and 2, primers 5 -GTTCAAGGGGATGGAGGAGC-3 and 5 TCATCATTCACCTTCTTCCCAA-3 [15] were introduced. The PCR was carried out in a LightCycler 2.0 instrument (Roche Diagnostics GmbH, Manheim, Germany) and consisted of an initial denaturation of 2 minutes at 95 ◦ C, followed by 35 cycles of 30 seconds at 95 ◦ C, 50 seconds at 60 ◦ C, 2 minutes at 72 ◦ C and a final elongation of 7 minutes at 72 ◦ C. A 25 L PCR mixture contained 1 × High Fidelity Enyzme buffer, 0.25 mol/L each primer, 0.2 mM dNTPs, 1U High Fidelity Expand enzyme (Roche Diagnostics GmbH, Manheim, Germany) and 10 ng of DNA. RHD and RHCE exons 1 to 10 including intron/exon boundaries were amplified as published before [16,17]. PCR products were treated with alkaline phosphatase and exonuclease I (ExoSAP-IT, High Wycombe, UK) before analyzing by an automated ABI 3130 sequencer (Life Technologies, Vienna, Austria) using BigDye Chemistry (BigDye Terminator v1.1 Cycle Sequencing Kit, Life Technologies). Data analysis was performed with SeqScape v. 2.7 (Life Technologies). 3. Results In this study, we verified six Fy phenotypes among 105 samples, comprising Fy(a-b+) (n = 40, 38.1%), Fy(a+b-) (n = 24, 22.9%), Fy(a+b+) (n = 32, 30.5%), Fy(a-b-) (n = 5, 4.8%), Fy(ab+w ) Fyx (n = 3, 2.9%), Fy(a+b+w ) Fyx (n = 1, 1%). Standard serological Fy typing revealed a diminished expression of the Fyb antigen in 4 samples and a Fy(a-b-) phenotype in 5 individuals (Table 1). Those samples underwent molecular characterization by nucleotide sequencing of the DARC gene. The FY*02M.01 gene formation was determined by DARC(Arg89Cys,Ala100Thr) in a homozygous (n = 2) and heterozygous occurrence (n = 1) (Table 2). In one sample with diminished Fyb phenotype, no alteration of the DARC gene Table 1 Results of serological Duffy typing. Duffy phenotype
n
%
Fy(a+b+) Fy(a+b-) Fy(a-b+) Fy(a-b-) Fy(a-b+w )Fyx Fy(a+b+w )Fyx
32 24 40 5 3 1
30.5 22.9 38.1 4.8 2.9 1.0
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Table 2 Molecular DARC, RHD and RHCE typing results of samples with aberrant Fy phenotype. Fy phenotype
FY genotype
RHD genotype
RHCE genotype
Fy(a-b-)
FY*02N.01/FY*02N.01 FY*02N.01/FY*02N.01 FY*02N.01/FY*02N.01 FY*02N.01/FY*02N.01 FY*02N.01/FY*02N.01 FY*A/FY*02M.01 FY*02M.01/FY*02M.01 FY*02M.01/FY*02M.01
RHD/RHD*01N.01 RHD*01N.01/RHD*01N.01 RHD/RHD*01N.01 RHD/RHD*01N.01 RHD/RHD*01N.01 RHD/RHD*01N.01 RHD*DAU-3/RHD*01N.01 RHD(165C>T)/RHD
RHCE*Ce/ceVS.02 RHCE*ce/ce01.06 RHCE*Ce/ce01.06 RHCE*Ce/Ce RHCE*Ce/ce RHCE*ce/ceVS.01 RHCE*Ce/ce RHCE*cE/Ce(165C>T, 486 C>T)
Fy(a+b+w ) Fyx Fy(a-b+w ) Fyx Fy(a-b+w ) Fyx
was determined. Further serological characterization revealed a positive DAT, accounting for the inconclusive Fy typing results. All 5 samples without expression of the Fy antigen were shown to harbour the 67C>T mutation in the GATA-1 binding motif of the FY*B promoter in a homozygous occurrence associated with a FYnull phenotype (Table 2). Molecular characterization of the RH genes revealed a RHD gene deletion in one sample associated with a RHCE*ce/ce01.06 allele encoded by RHCE(Ala85Gly). The other 4 individuals carried a common hemizygous RHD allele. The presence of RHCE*Ce/ceVS.02 was determined in one sample by RHCE(Trp16Cys,Leu245Val) (Table 2). One individual with a Fy(a+b+w ) phenotype exhibited a RHCE*ceVS.01 (Leu245Val) with RHCE*ce on the partner chromosome. No alterations were determined in its RHD gene sequence. Out of two individuals with Fy(a-b+w ), one was shown to exhibit a RHD*DAU-3 characterized by RHD(Val279Met, Thr379Met) and a RHCE*Ce/ce. The second sample harbored a heterozygous nucleotide alteration 165C>T in RHD exon 2. Interestingly, the RHCE allele of this sample also revealed the 165C>T mutation in a heterozygous occurrence. In addition, another silent nucleotide substitution was located in exon 3, RHCE(486C>T). No alteration in the RhCE expression was observed, probably due to a normal RHCE in trans. 4. Discussion The most common phenotype Fy(a-b+) occured with a frequency of 38.1% similar to that reported for Caucasians (34%), but much higher than that of 22% described for the Black population [9]. Among 105 Tunisian blood donor samples, 5 (4.8%) harbored a FYnull phenotype, caused by a homozygous FY*02N.01 genotype. This allele is uncommon in the Caucasian population, whereas it was described with a frequency of 68% in Blacks [9] and in 11.3% in a previous study of Tunisians blood donors [18]. The African sub-saharan genetic contribution in our cohort seems low than in the previous study [18]. This disparity may be explained by the different sampling area and the sampling size of our study. We not confirm the FY*01.N.01 previously described in our population [18], but the FY null phenotype described in this presented work is due to FY*02N.01 genotype which is frequently observed in the black population [8]. Standard serological Fy typing showed the presence of 4 samples with diminished expression of Fyb antigen Nucleotide
sequencing of these samples confirmed the FY*02M.01 allele in 3 individuals. Samples with a diminished expression or lack of the Fy antigen underwent DNA sequencing of RHD and RHCE exons 1-10. Not surprisingly, aberrant gene formations associated with Black populations were confirmed among those samples. The RHCE*ceVS.02 allele is encoded by two mutations, 48G>C and 733C>G, and leads to expression of V, VS and hrB [19]. In the black population, the 48G>C substitution is common in a ce haplotype [20]. The 733C>G substitution in RHCE exon 5 leads to the Leu245Val amino acid change and forces the expression of the VS antigen [19–21] which is extremely rare in people of European and Asian origin but has a frequency of up to 50% in African descents [19–21]. We suggest that for the Tunisian region, pre-transfusion testing should include an antibody screening test for the VS antigen. The RHCE*ce01.06 allele results from a single point mutation in RHCE exon 2 (254C>G) leading to a Ala85Gly substitution in the third transmembrane domain. It encodes partial e and showes the absence of the CEAG and hrB antigens [9]. In our case, the presence of a normal RHCE*ce in trans masked an aberrant RhCE expression. One sample with Fy(a-b+w ) was associated with RHD(165C>T)/RHD and RHCE*cE/Ce(165C>T, 486C>T). The RHD(165C>T) allele was annotated before to occur with a normal D in an Austrian patient (accession no. FN545815), a weakened D in a patient from China (accession no. DQ309583) and in trans to a normal D in one Tibetian individual (AM398143). Interestingly, the RHCE allele of this sample also revealed the 165C>T mutation in a heterozygous occurrence. In our study, this sample expressed a normal D phenotype. A possible weakened D antigen would have been masked by the normal RHD in trans. To our knowledge, the associated RHCE(165C>T, 486C>T) allele was not determined before. Both nucleotide alterations do not lead to an amino acid substitution, and we could not find any influence on the RhCE. Several studies have been undertaken to describe the genetic structure of Tunisian populations employing genetic markers specific for immunoglobulin chains [22], HLA class II [23], autosomal short tandem repeats (STRs) [24], and polymorphic Alu insertions [25]. Our findings underline the results of genetically based population studies which indicate that the Tunisian genetic distances to Europa are small [26]. This may be easily explained by the eventful history of the country concerning occupations and immigrations. The small sub-Saharan genetic
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