- Email: [email protected]
Molecular identification of rare FY*Null and FY*X alleles in Caucasian thalassemic family from Sardinia
ARTICLE IN PRESS Transfusion and Apheresis Science ■■ (2015) ■■–■■
Contents lists available at ScienceDirect
Transfusion and Apheresis Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a n s c i
Molecular identification of rare FY*Null and FY*X alleles in Caucasian thalassemic family from Sardinia Silvia Manfroi *, Antonio Scarcello 1, Pasqualepaolo Pagliaro 2 Immunohematology and Transfusion Service, Policlinico S.Orsola-Malpighi, Via Massarenti, 9, 40138 Bologna, Italy
A R T I C L E
I N F O
Article history: Received 3 November 2014 Received in revised form 3 April 2015 Accepted 8 April 2015 Keywords: Duffy blood group Positive selection FY*Null FY*X
A B S T R A C T
Molecular genetic studies on Duffy blood group antigens have identified mutations underlying rare FY*Null and FY*X alleles. FY*Null has a high frequency in Blacks, especially from sub-Saharan Africa, while its frequency is not defined in Caucasians. FY*X allele, associated with Fy(a-b + w) phenotype, has a frequency of 2–3.5% in Caucasian people while it is absent in Blacks. During the project of extensive blood group genotyping in patients affected by hemoglobinopathies, we identified FY*X/FY*Null and FY*A/FY*Null genotypes in a Caucasian thalassemic family from Sardinia. We speculate on the frequency of FY*X and FY*Null alleles in Caucasian and Black people; further, we focused on the association of FY*X allele with weak Fyb antigen expression on red blood cells and its identification performing high sensitivity serological typing methods or genotyping. © 2015 Published by Elsevier Ltd.
1. Introduction The DARC (Duffy blood group, chemokine receptor) gene encodes an integral membrane glycoprotein which expresses Duffy blood group antigens and functions both as a chemokine transporter and as a receptor for the malaria parasites Plasmodium vivax and Plasmodium knowlesi [1,2]. The Duffy blood group system includes two antithetical antigens, Fya and Fyb. These antigens are encoded by codominant alleles FY*A (FY*01) and FY*B (FY*02), which differ by a single point mutation, defining Fy(a + b-), Fy(ab+) and Fy(a + b+) phenotypes. The molecular mechanism that gives rise to the null Duffy phenotype Fy(a-b-) has been identified in GATA-1 transcription factor binding motif of promoter region of FY*B allele, which codes for the Fyb antigen. The GATA-1 mutation is in the FY*Null (FY*02N.01)
* Corresponding author. Servizio Immunoematologia e Trasfusionale, Policlinico S.Orsola-Malpighi, Via Massarenti, 9, 40138 Bologna, Italy. Tel.: +39 051 6363901; fax: +39 051 6363527. E-mail address: [email protected] (S. Manfroi). 1 Permanent address: Via Antonio Lanzone, 2, Torano Castello, Cosenza, Italy. 2 Permanent address: Via Santa Croce, 20, Milano, Italy.
allele, which has the same coding sequence as the FY*B allele. A point mutation (T > C) at nucleotide -67 impairs the promoter activity and suppresses Duffy antigen expression in bone marrow derived cells but not in other tissues. The absence of Duffy antigens on red blood cells (RBC) prevents their invasion by Plasmodium vivax and Plasmodium knowlesi [1,3]. The Fy(a-b + w) phenotype is associated with weak Fyb expression and it depends on two mutations in the coding region of FY*B gene (265 C > T and 298G>A). These mutations encode amino acid substitutions Arg89Cys and Ala100Thr, respectively, and give rise to FY*X(FY*02M.01) allele. 265 C > T substitution is the critical mutation responsible for an unstable protein and (due to altered posttranslational processing) for reduced Fyb antigen expression on red cell surface. 298G>A mutation (encoding Ala100Thr), which occurs by itself at a frequency of 27–33% in white donors, does not affect Fyb antigen expression. Homozygosis for FY*Null allele is responsible for Fy(a-b-) phenotype: while it is very rare in Caucasian people, the frequency of the null phenotype differs in different populations of Black individuals, reaching 100% in some. The FY*X allele has a frequency of 2–3.5% in Caucasians and is absent in Blacks [4–6].
http://dx.doi.org/10.1016/j.transci.2015.04.013 1473-0502/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Silvia Manfroi, Antonio Scarcello, Pasqualepaolo Pagliaro, Molecular identification of rare FY*Null and FY*X alleles in Caucasian thalassemic family from Sardinia, Transfusion and Apheresis Science (2015), doi: 10.1016/j.transci.2015.04.013
ARTICLE IN PRESS S. Manfroi et al./Transfusion and Apheresis Science ■■ (2015) ■■–■■
2
Table 1 Duffy serological and molecular typing. Phenotype
Genotype
Tube test
Microcolumn test
PCR-SSP
BLOODChip1
Predicted phenotype1
Patient
Fy(a + b-)
Fy(a + b-)
FY*A/Null
Fya, Fy null
Mother Father
Fy(a + b+) Fy(a-b-)
Fy(a + b+) Fy(a-b + w)
FY*A/B FY*X/Null
FY*A/FY*B FY* GATA-1 het1 FY*A/FY*B1 FY*B/FY*B FY*GATA-1 het FY*X-het1
1
Fya, Fyb Fyb weak
As indicated in BIDS analysis report.
This report describes the case of a Caucasian thalassemic family in which we identified Fy(a-b + w) and Fy(a + b-) phenotypes with, respectively, FY*X/Null and FY*A/Null genotypes. The identification was performed within the project of extensive blood group genotyping in patients affected by hemoglobinopathies (sickle cell disease, β-thalassemia) with the aim of providing them with better-matched compatible blood products. 2. Materials and methods We performed genotyping on DNA samples from 99 adult and pediatric patients (89 Caucasians, 10 Black Africans) affected by various hemoglobinopathies and hematological disorders: β-thalassemia (n = 81), sickle cell disease (SCD) (n = 12), microdrepanocytosis (n = 3), Blackfan–Diamond anemia (n = 2), and aplastic anemia (n = 1). DNA isolated from buffy coat samples was genotyped using BLOODchip® IDCore+ (Progenika-Grifols, Spain). Results were analyzed by xMAP Luminex® technology (Luminex Corporation, USA) and BLOODchip® ID software (BIDS) (Progenika-Grifols, Spain). Confirmatory typing of the genotype was performed by PCR-SSP (RBC-Ready Gene KKD, Innotrain, Germany). RBC serology typing was performed by antiglobulin test with human polyclonal anti-Fya and anti-Fyb antisera (Immucor, Germany), with tube and microcolumn test (ID-Card, Diamed, Switzerland), according to manufacturers’ instructions. Weak expression of the Fyb antigen was confirmed by adsorption/elution method using Gamma Elu-Kit II (Immucor, Germany). 3. Results The Fy(a–b–) phenotype (FY*Null/Null genotype) was identified in all the patients of African descent (10/89): they have SCD (n = 9) and microdrepanocytosis (n = 1) diagnosis. The FY*Null allele was identified in a Caucasian female, 35 years old, affected by homozygous β-thalassemia. The sample, genotyped on IDCORE+, resulted FY*A/B, no FY*X and heterozygous for FY GATA-1 mutation: this means that there were coding sequences for both FY*A and FY*B alleles, but only Fya antigen would be translated and expressed on erythrocyte surface. BIDS software warned to notice the genotype result and correctly converted it in the predicted Fy(a + b-) phenotype, concordant with serological typing. Test repetition confirmed the genotyping results and confirmatory
PCR-SSP was performed: the patient genotype was found to be FY*A/Null. The patient is a resident in the Italian region of Emilia Romagna, but her family comes from Sardinia, which is the Mediterranean region with the highest incidence of β–thalassemia (11% frequency) and, in the past, was endemic for malaria. Genetic family study was performed on parents, both with β-thalassemia trait, using IDCORE+ and confirmatory PCR-SSP. The mother sample was genotyped as FY*A/B and the predicted phenotype, Fy(a + b+), was concordant with serological typing. The father’s genotype was FY*B/B, and heterozygous for FY*X and FY GATA-1 mutation (confirmed by PCR-SSP). The predicted phenotype was Fy(a-b + w) and it was concordant with serological typing, which showed a marked decrease in anti-Fyb agglutinability; Fyb was detected only in LISS/Coombs microcolumn but not with tube test. Adsorption with anti-Fyb showed a reactive eluate. We could not genotype other family members because the parents are the only ones alive (Table 1). 4. Discussion FY*X allele has been described mainly among Caucasians. Studies conducted on Northern European and Canadian populations [4,5] found a frequency varying from 2% to 3.5%; the FY*X allele was not described in Blacks. Recently, Schmid et al. [7], developing a phylogenetic tree for DARC alleles, sequenced only one FY*X allele out of 54 African American blood donors, and the authors considered 265C>T mutation as the most recently acquired polymorphism. On the contrary, negative Duffy phenotype has a wide distribution in sub-Saharan Africa (65% to almost 100%) and in localized areas of the Americas. It is well known that Duffy negativity (due to FY*Null/Null genotype) is responsible for high-level resistance to Plasmodium vivax blood-stage infection [8,9]. In a recent study on global distribution of the Duffy blood group [10], 131,187 individuals from four continents were typed for Duffy antigens: FY*Null/Null genotype was identified in two samples out of 37,457 Europeans and none revealed a FY*A/Null genotype, whereas the last genotype had a frequency of 13% in African and of 11% in American population; FY*X allele was not investigated. In another study of Duffy genotype and allele frequencies in about 100 individuals of black South African, white Swedish and Han Chinese populations respectively [11], FY*A/Null genotype
Please cite this article in press as: Silvia Manfroi, Antonio Scarcello, Pasqualepaolo Pagliaro, Molecular identification of rare FY*Null and FY*X alleles in Caucasian thalassemic family from Sardinia, Transfusion and Apheresis Science (2015), doi: 10.1016/j.transci.2015.04.013
ARTICLE IN PRESS S. Manfroi et al./Transfusion and Apheresis Science ■■ (2015) ■■–■■
had a frequency of 4% in Blacks and it was not found in White and Chinese individuals. Hashmi et al. reported the frequency of FY*A or FYB/FY*Null in Caucasian American Blood donors to be 0.02 [12]. The FY*A/Null genotype identified in the patient is confirmed very rare in our population as well as the father’s genotype (FY*X/Null): 1 out 89 tested patients (1,1%). The members of the family reported that their great grandparents originated from the southern area of Sardinia, without having knowledge of any ancestor from Africa in the past. Data on classical genetic markers showed a high level of internal heterogeneity and a complex relationship between Sardinia and other Italian and Mediterranean populations. A study based on the frequencies of several classical genetic markers (phosphoglucomutase1 (PGM1), MN and RH alleles) [13] shows some of the distinctive features that set Sardinians apart from other Mediterranean populations. At the same time, the study of HLA genes suggests important contacts between Sardinia and northern Africa [14]. Unfortunately, population studies for Caucasian population in Sardinia using FY*Null allele are not available. Actually, the present genetic composition of Sardinians is determined by the contributions of several ethnic groups of north-western Mediterranean, eastern Mediterranean and northern African origins inserted on a palaeo-Mediterranean substrate [15], as suggested by the analysis of DNA markers, mtDNA and Y chromosome. Further, some geographic, genetic and linguistic observations suggest that a possible path for the settlement of the island by Homo sapiens started from northern Africa. The hypothesis is that Sardinia would have been therefore a gateway between Africa and Europe, and small groups would have moved bidirectionally through the island. Sardinia was in the past endemic for malaria, but the epidemiology of the disease in the island is rather complex because both Plasmodium vivax and Plasmodium falciparum coexisted, neither strain was predominant and multiple infections were not uncommon. In general, Plasmodium vivax had a consistently high annual incidence which accounted for the stable, endemic nature of malaria in Sardinia and was characterized by high morbidity but low mortality rates. On the other hand, Plasmodium falciparum, the more deadly strain, had a more seasonal nature and accounts for the annual epidemic during late summer, depending upon variable ecological conditions such as the amount of summer precipitation [16,17]. The selective advantage on malaria infection conferred by β-thalassemia is well known, which has 11% of frequency in Sardinia, as well as the strong advantage to having Fy(ab-) phenotype, as in people of African origin. In our brief experience in RBC genotyping, FY*Null allele has a low frequency (1.1%) in Caucasian patients and its presence in the patient genotype could be explained by a population migration of unknown ancient forefather from Africa northern coast, not far from southern area of Sardinia, the area of origin of the patient’s family. If the FY*X allele had been a selective advantage, it would probably have been more common. Although mistyping of Fyb antigen can easily depend on the anti-Fyb reagent used [18], weak Fyb expression can be accounted by heterozygous GATA box mutation (RBCs have one-half of the level of wildtype FY*B/B RBCs) or Fy(b + w)
3
phenotype (RBCs have about one-tenth of the level) [5]. Some authors suggest to perform FY genotyping of blood donors to exclude the possibility of the heterozygous FY*X allele and to assure the true absence of Fyb antigen in Fyb negative blood [4,5]. Nevertheless, transfusion of Fy(b + w) blood units to a Fyb negative patient or to a patient with an anti-Fyb is not known to cause primary alloimmunization or hemolytic transfusion reaction [4]. Meanwhile, because FY*X-associated mutations cause a reduced Fyb antigen expression on RBC surface without evidence of conformational changes in the protein, FY*X patients could potentially receive Fyb positive RBC, increasing available blood donors and saving Fyb negative blood. Obviously FY genotyping, as part of extended genomic RBC typing, should be performed in chronically transfused patients or at increased risk of alloantibody formation.
References [1] Meny GM. The Duffy blood group system: a review. Immunohematology 2010;26:51–6. [2] Rowe JA, Opi DH, Williams TN. Blood groups and malaria: fresh insights into pathogenesis and identification of targets for intervention. Curr Opin Hematol 2009;16:480–7. [3] Tournamille C, Colin Y, Cartron 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. [4] Olsson ML, Smythe JS, Hansson C, Poole J, Mallinson G, Jones J, et al. The Fyx phenotype is associated with a missense mutation in the Fyb allele predicting Arg89Cys in the Duffy glycoprotein. Br J Haematol 1998;103:1184–91. [5] Yazdanbakhsh K, Rios M, Storry JR, Kosower N, Parasol N, Chaudhuri A, et al. Molecular mechanisms that lead to reduced expression of Duffy antigens. Transfusion 2000;40:310–20. [6] Daniels J. Duffy blood group system. In: Human blood groups. 3rd ed. Oxford: Wiley-Blackwell; 2013. p. 306–24. [7] Schmid P, Ravenell KR, Sheldon SR, Flegel WA. DARC alleles and Duffy phenotypes in African Americans. Transfusion 2012;52:1260–7. [8] Miller LH, Mason SJ, Clyde DF, McGinniss MH. The resistance factor to Plasmodium vivax in Blacks. The Duffy blood group phenotype, FyFy. N Engl J Med 1976;295:302–4. [9] King CL, Adams JH, Xianli J, Grimberg BT, McHenry AM, Greenberg LJ, et al. FYa/Fyb antigen polymorphism in human erythrocyte Duffy antigen affects susceptibility to Plasmodium vivax malaria. Proc Natl Acad Sci U S A 2011;108:20113–8. [10] Howes RE, Patil AP, Piel FB, Nyangiri OA, Kebaria CW, Gething PW, et al. The global distribution of the Duffy blood group. Nat Commun 2011;2:266. doi:10.1038/ncomms1265. [11] Olsson ML, Hansson C, Avent ND, Akesson IE, Green CA, Daniels GL. A clinically applicable method for determining the three major alleles at the Duffy (FY) blood group locus using polymerase chain reaction with allele-specific primers. Transfusion 1998;38:168–73. [12] Hashmi G, Shariff T, Zhang Y, Cristobal J, Chau C, Seul M, et al. Determination of 24 minor red blood cell antigens for more than 2000 blood donors by high-throughput DNA analysis. Transfusion 2007;47:736–47. [13] Moral P, Marogna G, Salis M, Succa V, Vona G. Genetic data on Alghero population (Sardinia): contrast between biological and cultural evidence. Am J Phys Anthropol 1994;93:441–53. [14] Grimaldi MC, Crouau-Roy B, Amoros JP, Cambon-Thomsen A, Carcassi C, Orru S, et al. West Mediterranean islands (Corsica, Balearic islands, Sardinia) and the Basque population: contribution of HLA class I molecular markers to their evolutionary history. Tissue Antigens 2001;58:281–92. [15] Piazza A, van Loghem E, de Lange G, Curtoni ES, Ulizzi L, Terrenato L. Immunoglobulin allotypes in Sardinia. Am J Hum Genet 1976;28:77–86. [16] Aitken T. The anopheline fauna of Sardinia. Am J Hyg 1953;20:303–52. [17] Hackett LW. Malaria in Europe: an ecological study. London: Oxford University Press; 1937. [18] Murphy MT, Templeton LJ, Fleming J, Ferguson M, Peterkin M, Fraser RH. Comparison of Fy b status as determined serologically and genetically. Transfus Med 1997;7:135–41.
Please cite this article in press as: Silvia Manfroi, Antonio Scarcello, Pasqualepaolo Pagliaro, Molecular identification of rare FY*Null and FY*X alleles in Caucasian thalassemic family from Sardinia, Transfusion and Apheresis Science (2015), doi: 10.1016/j.transci.2015.04.013
Contents lists available at ScienceDirect
Transfusion and Apheresis Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a n s c i
Molecular identification of rare FY*Null and FY*X alleles in Caucasian thalassemic family from Sardinia Silvia Manfroi *, Antonio Scarcello 1, Pasqualepaolo Pagliaro 2 Immunohematology and Transfusion Service, Policlinico S.Orsola-Malpighi, Via Massarenti, 9, 40138 Bologna, Italy
A R T I C L E
I N F O
Article history: Received 3 November 2014 Received in revised form 3 April 2015 Accepted 8 April 2015 Keywords: Duffy blood group Positive selection FY*Null FY*X
A B S T R A C T
Molecular genetic studies on Duffy blood group antigens have identified mutations underlying rare FY*Null and FY*X alleles. FY*Null has a high frequency in Blacks, especially from sub-Saharan Africa, while its frequency is not defined in Caucasians. FY*X allele, associated with Fy(a-b + w) phenotype, has a frequency of 2–3.5% in Caucasian people while it is absent in Blacks. During the project of extensive blood group genotyping in patients affected by hemoglobinopathies, we identified FY*X/FY*Null and FY*A/FY*Null genotypes in a Caucasian thalassemic family from Sardinia. We speculate on the frequency of FY*X and FY*Null alleles in Caucasian and Black people; further, we focused on the association of FY*X allele with weak Fyb antigen expression on red blood cells and its identification performing high sensitivity serological typing methods or genotyping. © 2015 Published by Elsevier Ltd.
1. Introduction The DARC (Duffy blood group, chemokine receptor) gene encodes an integral membrane glycoprotein which expresses Duffy blood group antigens and functions both as a chemokine transporter and as a receptor for the malaria parasites Plasmodium vivax and Plasmodium knowlesi [1,2]. The Duffy blood group system includes two antithetical antigens, Fya and Fyb. These antigens are encoded by codominant alleles FY*A (FY*01) and FY*B (FY*02), which differ by a single point mutation, defining Fy(a + b-), Fy(ab+) and Fy(a + b+) phenotypes. The molecular mechanism that gives rise to the null Duffy phenotype Fy(a-b-) has been identified in GATA-1 transcription factor binding motif of promoter region of FY*B allele, which codes for the Fyb antigen. The GATA-1 mutation is in the FY*Null (FY*02N.01)
* Corresponding author. Servizio Immunoematologia e Trasfusionale, Policlinico S.Orsola-Malpighi, Via Massarenti, 9, 40138 Bologna, Italy. Tel.: +39 051 6363901; fax: +39 051 6363527. E-mail address: [email protected] (S. Manfroi). 1 Permanent address: Via Antonio Lanzone, 2, Torano Castello, Cosenza, Italy. 2 Permanent address: Via Santa Croce, 20, Milano, Italy.
allele, which has the same coding sequence as the FY*B allele. A point mutation (T > C) at nucleotide -67 impairs the promoter activity and suppresses Duffy antigen expression in bone marrow derived cells but not in other tissues. The absence of Duffy antigens on red blood cells (RBC) prevents their invasion by Plasmodium vivax and Plasmodium knowlesi [1,3]. The Fy(a-b + w) phenotype is associated with weak Fyb expression and it depends on two mutations in the coding region of FY*B gene (265 C > T and 298G>A). These mutations encode amino acid substitutions Arg89Cys and Ala100Thr, respectively, and give rise to FY*X(FY*02M.01) allele. 265 C > T substitution is the critical mutation responsible for an unstable protein and (due to altered posttranslational processing) for reduced Fyb antigen expression on red cell surface. 298G>A mutation (encoding Ala100Thr), which occurs by itself at a frequency of 27–33% in white donors, does not affect Fyb antigen expression. Homozygosis for FY*Null allele is responsible for Fy(a-b-) phenotype: while it is very rare in Caucasian people, the frequency of the null phenotype differs in different populations of Black individuals, reaching 100% in some. The FY*X allele has a frequency of 2–3.5% in Caucasians and is absent in Blacks [4–6].
http://dx.doi.org/10.1016/j.transci.2015.04.013 1473-0502/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Silvia Manfroi, Antonio Scarcello, Pasqualepaolo Pagliaro, Molecular identification of rare FY*Null and FY*X alleles in Caucasian thalassemic family from Sardinia, Transfusion and Apheresis Science (2015), doi: 10.1016/j.transci.2015.04.013
ARTICLE IN PRESS S. Manfroi et al./Transfusion and Apheresis Science ■■ (2015) ■■–■■
2
Table 1 Duffy serological and molecular typing. Phenotype
Genotype
Tube test
Microcolumn test
PCR-SSP
BLOODChip1
Predicted phenotype1
Patient
Fy(a + b-)
Fy(a + b-)
FY*A/Null
Fya, Fy null
Mother Father
Fy(a + b+) Fy(a-b-)
Fy(a + b+) Fy(a-b + w)
FY*A/B FY*X/Null
FY*A/FY*B FY* GATA-1 het1 FY*A/FY*B1 FY*B/FY*B FY*GATA-1 het FY*X-het1
1
Fya, Fyb Fyb weak
As indicated in BIDS analysis report.
This report describes the case of a Caucasian thalassemic family in which we identified Fy(a-b + w) and Fy(a + b-) phenotypes with, respectively, FY*X/Null and FY*A/Null genotypes. The identification was performed within the project of extensive blood group genotyping in patients affected by hemoglobinopathies (sickle cell disease, β-thalassemia) with the aim of providing them with better-matched compatible blood products. 2. Materials and methods We performed genotyping on DNA samples from 99 adult and pediatric patients (89 Caucasians, 10 Black Africans) affected by various hemoglobinopathies and hematological disorders: β-thalassemia (n = 81), sickle cell disease (SCD) (n = 12), microdrepanocytosis (n = 3), Blackfan–Diamond anemia (n = 2), and aplastic anemia (n = 1). DNA isolated from buffy coat samples was genotyped using BLOODchip® IDCore+ (Progenika-Grifols, Spain). Results were analyzed by xMAP Luminex® technology (Luminex Corporation, USA) and BLOODchip® ID software (BIDS) (Progenika-Grifols, Spain). Confirmatory typing of the genotype was performed by PCR-SSP (RBC-Ready Gene KKD, Innotrain, Germany). RBC serology typing was performed by antiglobulin test with human polyclonal anti-Fya and anti-Fyb antisera (Immucor, Germany), with tube and microcolumn test (ID-Card, Diamed, Switzerland), according to manufacturers’ instructions. Weak expression of the Fyb antigen was confirmed by adsorption/elution method using Gamma Elu-Kit II (Immucor, Germany). 3. Results The Fy(a–b–) phenotype (FY*Null/Null genotype) was identified in all the patients of African descent (10/89): they have SCD (n = 9) and microdrepanocytosis (n = 1) diagnosis. The FY*Null allele was identified in a Caucasian female, 35 years old, affected by homozygous β-thalassemia. The sample, genotyped on IDCORE+, resulted FY*A/B, no FY*X and heterozygous for FY GATA-1 mutation: this means that there were coding sequences for both FY*A and FY*B alleles, but only Fya antigen would be translated and expressed on erythrocyte surface. BIDS software warned to notice the genotype result and correctly converted it in the predicted Fy(a + b-) phenotype, concordant with serological typing. Test repetition confirmed the genotyping results and confirmatory
PCR-SSP was performed: the patient genotype was found to be FY*A/Null. The patient is a resident in the Italian region of Emilia Romagna, but her family comes from Sardinia, which is the Mediterranean region with the highest incidence of β–thalassemia (11% frequency) and, in the past, was endemic for malaria. Genetic family study was performed on parents, both with β-thalassemia trait, using IDCORE+ and confirmatory PCR-SSP. The mother sample was genotyped as FY*A/B and the predicted phenotype, Fy(a + b+), was concordant with serological typing. The father’s genotype was FY*B/B, and heterozygous for FY*X and FY GATA-1 mutation (confirmed by PCR-SSP). The predicted phenotype was Fy(a-b + w) and it was concordant with serological typing, which showed a marked decrease in anti-Fyb agglutinability; Fyb was detected only in LISS/Coombs microcolumn but not with tube test. Adsorption with anti-Fyb showed a reactive eluate. We could not genotype other family members because the parents are the only ones alive (Table 1). 4. Discussion FY*X allele has been described mainly among Caucasians. Studies conducted on Northern European and Canadian populations [4,5] found a frequency varying from 2% to 3.5%; the FY*X allele was not described in Blacks. Recently, Schmid et al. [7], developing a phylogenetic tree for DARC alleles, sequenced only one FY*X allele out of 54 African American blood donors, and the authors considered 265C>T mutation as the most recently acquired polymorphism. On the contrary, negative Duffy phenotype has a wide distribution in sub-Saharan Africa (65% to almost 100%) and in localized areas of the Americas. It is well known that Duffy negativity (due to FY*Null/Null genotype) is responsible for high-level resistance to Plasmodium vivax blood-stage infection [8,9]. In a recent study on global distribution of the Duffy blood group [10], 131,187 individuals from four continents were typed for Duffy antigens: FY*Null/Null genotype was identified in two samples out of 37,457 Europeans and none revealed a FY*A/Null genotype, whereas the last genotype had a frequency of 13% in African and of 11% in American population; FY*X allele was not investigated. In another study of Duffy genotype and allele frequencies in about 100 individuals of black South African, white Swedish and Han Chinese populations respectively [11], FY*A/Null genotype
Please cite this article in press as: Silvia Manfroi, Antonio Scarcello, Pasqualepaolo Pagliaro, Molecular identification of rare FY*Null and FY*X alleles in Caucasian thalassemic family from Sardinia, Transfusion and Apheresis Science (2015), doi: 10.1016/j.transci.2015.04.013
ARTICLE IN PRESS S. Manfroi et al./Transfusion and Apheresis Science ■■ (2015) ■■–■■
had a frequency of 4% in Blacks and it was not found in White and Chinese individuals. Hashmi et al. reported the frequency of FY*A or FYB/FY*Null in Caucasian American Blood donors to be 0.02 [12]. The FY*A/Null genotype identified in the patient is confirmed very rare in our population as well as the father’s genotype (FY*X/Null): 1 out 89 tested patients (1,1%). The members of the family reported that their great grandparents originated from the southern area of Sardinia, without having knowledge of any ancestor from Africa in the past. Data on classical genetic markers showed a high level of internal heterogeneity and a complex relationship between Sardinia and other Italian and Mediterranean populations. A study based on the frequencies of several classical genetic markers (phosphoglucomutase1 (PGM1), MN and RH alleles) [13] shows some of the distinctive features that set Sardinians apart from other Mediterranean populations. At the same time, the study of HLA genes suggests important contacts between Sardinia and northern Africa [14]. Unfortunately, population studies for Caucasian population in Sardinia using FY*Null allele are not available. Actually, the present genetic composition of Sardinians is determined by the contributions of several ethnic groups of north-western Mediterranean, eastern Mediterranean and northern African origins inserted on a palaeo-Mediterranean substrate [15], as suggested by the analysis of DNA markers, mtDNA and Y chromosome. Further, some geographic, genetic and linguistic observations suggest that a possible path for the settlement of the island by Homo sapiens started from northern Africa. The hypothesis is that Sardinia would have been therefore a gateway between Africa and Europe, and small groups would have moved bidirectionally through the island. Sardinia was in the past endemic for malaria, but the epidemiology of the disease in the island is rather complex because both Plasmodium vivax and Plasmodium falciparum coexisted, neither strain was predominant and multiple infections were not uncommon. In general, Plasmodium vivax had a consistently high annual incidence which accounted for the stable, endemic nature of malaria in Sardinia and was characterized by high morbidity but low mortality rates. On the other hand, Plasmodium falciparum, the more deadly strain, had a more seasonal nature and accounts for the annual epidemic during late summer, depending upon variable ecological conditions such as the amount of summer precipitation [16,17]. The selective advantage on malaria infection conferred by β-thalassemia is well known, which has 11% of frequency in Sardinia, as well as the strong advantage to having Fy(ab-) phenotype, as in people of African origin. In our brief experience in RBC genotyping, FY*Null allele has a low frequency (1.1%) in Caucasian patients and its presence in the patient genotype could be explained by a population migration of unknown ancient forefather from Africa northern coast, not far from southern area of Sardinia, the area of origin of the patient’s family. If the FY*X allele had been a selective advantage, it would probably have been more common. Although mistyping of Fyb antigen can easily depend on the anti-Fyb reagent used [18], weak Fyb expression can be accounted by heterozygous GATA box mutation (RBCs have one-half of the level of wildtype FY*B/B RBCs) or Fy(b + w)
3
phenotype (RBCs have about one-tenth of the level) [5]. Some authors suggest to perform FY genotyping of blood donors to exclude the possibility of the heterozygous FY*X allele and to assure the true absence of Fyb antigen in Fyb negative blood [4,5]. Nevertheless, transfusion of Fy(b + w) blood units to a Fyb negative patient or to a patient with an anti-Fyb is not known to cause primary alloimmunization or hemolytic transfusion reaction [4]. Meanwhile, because FY*X-associated mutations cause a reduced Fyb antigen expression on RBC surface without evidence of conformational changes in the protein, FY*X patients could potentially receive Fyb positive RBC, increasing available blood donors and saving Fyb negative blood. Obviously FY genotyping, as part of extended genomic RBC typing, should be performed in chronically transfused patients or at increased risk of alloantibody formation.
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Please cite this article in press as: Silvia Manfroi, Antonio Scarcello, Pasqualepaolo Pagliaro, Molecular identification of rare FY*Null and FY*X alleles in Caucasian thalassemic family from Sardinia, Transfusion and Apheresis Science (2015), doi: 10.1016/j.transci.2015.04.013