Allelic polymorphisms of human platelets- specific ...

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of HPA-1, -3, and -5 systems in south Tunisian population, in order to ... Our study concerned 212 unrelated healthy, regular blood donors from southern Tunisia.
Original research paper

Allelic polymorphisms of human plateletsspecific alloantigens in South Tunisian population Mariem Smaoui 1, Basma HadjKacem2 , Ikram Ben Amor 1, Héla Mnif 1, Lobna Maalej 1, Ali Gargouri 2, Jalel Gargouri 1 1

Centre Régional de Transfusion Sanguine de Sfax, Université de Sfax, Sfax, Tunisia, 2Laboratoire de valorisation de la biomasse et production des protéines chez les eucaryotes, Centre de Biotechnologie de Sfax, Université de Sfax, Sfax, Tunisia Objectives: Human platelet-specific alloantigens (HPA) are polymorphic epitopes which vary among ethnic groups. Background: In Tunisia, HPA frequencies were determined in North and centre; however, the pattern of HPA in South Tunisian population is not been studied yet. The aim of this work was to determine allelic frequencies of HPA-1, -3, and -5 systems in south Tunisian population, in order to estimate the risk of anti-platelet alloimmunization and to create a register of HPA-typed blood donors. Methods: Our study concerned 212 unrelated healthy, regular blood donors from southern Tunisia. Allelic polymorphisms of each system were determined using a polymerase chain reaction with sequencespecific primers. Results: Genotype frequencies a/a, a/b, and b/b were, respectively, 0.670, 0.288, and 0.042 for HPA-1 system, 0.430, 0.462, and 0.108 for HPA-3 system, and 0.750, 0.241, and 0.009 for HPA-5 system. The allele frequencies were 0.814 and 0.186 for HPA-1a and -1b alleles; 0.660 and 0.340 for HPA-3a and -3b alleles and 0.870, and 0.130 for HPA-5a and -5b alleles. Discussion: The reported frequencies are more similar to those of Caucasians than those of north Tunisian population. Keywords: Human platelets antigens, PCR-SSP, Allelic polymorphism, Gene frequency, Tunisian population

Introduction Human platelet-specific alloantigens (HPA) are polymorphic epitopes of platelet membrane glycoproteins (GP).1 To date, 24 platelet-specific alloantigens have been defined by immune sera, of which 12 are grouped into six bi-allelic systems (HPA-1, -2, -3, -4, -5, and -15). The molecular basis of 22 of the 24 serologically defined antigens has been identified. For 21 platelet allo-antigens, the difference between self and non-self is distinguished by a single amino acid substitution, caused by a single-nucleotide polymorphism (SNP) in the gene encoding the relevant GP membrane.2 These epitopes are the target of platelet alloantibodies which can appear during pregnancy or after blood Correspondence to: Basma Hadjkacem, Laboratoire de valorisation et production de la biomasse chez lez eucaryotes, Centre de Biotechnologie de Sfax, BP’K’3038 Sfax, Tunisia. [email protected] Mariem Smaoui and Basma Hadjkacem are co-first authors.

© W. S. Maney & Son Ltd 2013 DOI 10.1179/1607845413Y.0000000087

transfusion.1 HPA allo-immunization is commonly responsible of three important clinical manifestations: Neonatal allo-immune thrombocytopenia (NAIT), post-transfusional purpura (PTP), and platelet transfusion refractoriness (PTR).3,4 The relative importance of each HPA system in antiplatelet allo-immunization varies among ethnic groups.5–7 Thus, the study of HPA polymorphism in different populations presents several interests. The first one is epidemiologically important in clinical practice and particularly the prediction of alloimmune disease.8 The second one gives support allo-immunized patients by the determination of HPA phenotypes and anti-HPA antibodies in order to allow the diagnosis, establish a prognosis, and to provide compatible platelets when needed.9,10 Finally, these studies present a genetic anthropological interest.8 Serological HPA phenotyping is very limited while platelet genotyping by molecular approach is widely

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Table 1

Sequences and length of designed primers for HPA-1, -3, -5 and the two internal positive control primers

Primer code HPA-1a-I HPA-1b-I HPA-1-II (common) HPA-3a-I HPA-3b-I HPA-3-II (common) HPA-5a-I HPA-5b-I HPA-5-II (common) CRP-I CRP-II

Primer sequences

Length primers

PCR products length (bp)

ACTTACAGGCCCTGCCTCT ACTTACAGGCCCTGCCTCC GTGCAATCCTCTGGGGACT GGGGGAGGGGCTGGGGA GGGGGAGGGGCTGGGGC GGCCCTGGGACTGTGAATG AGGAAGAGTCTACCTGTTTACTATCAAAG AGGAAGAGTCTACCTGTTTACTATCAAAA CTCTCATGGAAAATGGCAGTA CCAGCCTCTCTCATGCTTTTGGCCAGACAG GGGTCGAGGACAGTTCCGTGTAGAAGTGGA

19 19 19 17 17 19 29 29 21 30 30

189

293

256

440

I: forward primer; II: reverse primer

recommended. Several techniques for SNP typing have been described for platelet genotyping. Polymerase chain reaction (PCR) amplification with sequence-specific primers (PCR-SSP) is currently used.7 The aim of this work was to determinate the allelic frequencies of HPA-1, -3, and -5 systems, in order to estimate the risk of anti-platelet allo-immunization and to create a register of HPA-typed blood donors in the south of Tunisia.

Materials and methods Blood donors, samples, and gene amplification

1

2

This study concerned 212 blood donors (148 men and 64 women). All of them are unrelated regular healthy volunteers, from southern Tunisia (Sfax City: n =172, Delegations of Sfax and South: n = 40). They were divided into 147 whole-blood donors and 65 platelets donors. Genomic DNA was extracted by the saltingout method,11 from venous blood samples of 10 ml each collected on EDTA. HPA-1, -3, and -5 systems were genotyped by PCRSSP. The sequences of primers used here are according to Klüter et al. 7 A pair of primers amplifying a conserved region of the gene for C-reactive protein (CRP) was included in each tube, as internal control (Table 1). PCR was performed as described previously by Klüter et al. 7 using a Gene Amp PCR System 9700, Applied Biosystem Thermal Cycler, in a volume of 20 μl. Positive and negative controls were included in each series. PCR products were visualized by UV trans-illumination after electrophoresis on agarose gel at 1–2% and staining with ethidium bromide.

Statistical analysis Genotype frequencies were determined by direct counting. Allele frequencies were calculated from genotype frequencies. The confidence interval was set at 5%. The validity of Hardy–Weinberg equilibrium for each of the HPA system was tested by χ 2 test.12 2

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Comparisons of allele frequencies between different populations were assessed by the χ 2 test (reduced gap). The significance level was chosen to be 5%.

Results In our study, PCR conditions were set different for HPA-1b, -3b, -5a, and -5b alleles. In fact, the alleles of the same system have different hybridization temperatures. Our work was therefore organized in several series of blood donors for each allele. We have chosen nine HPA-specific primers and two CRP primers (internal positive control) for HPA-1, -3, and -5 systems genotyping. We used also DNAs with known HPA genotypes. In Fig. 1, we give an example of the results obtained by the PCR-SSP method showing that primers specifically amplify the desired platelet HPA-1a-related DNA and that the two alloantigen-specific primers clearly distinguish between the two alleles. The 440 bp amplification product of CRP control primer was present in all

Figure 1 Examples of amplification in HPA-1a system. The upper band corresponds to the amplification product of the CRP gene (internal control of 440 bp). The lower band corresponds to the amplification product of HPA-1a gene (189 bp) situated between 123 and 246 bp bands of molecular weight markers.Lane1: negative control; lanes 2, 3, 4, 6, and 7: individuals with HPA-1a allele; lane 5: individual without HPA1a allele; lane 8: molecular weight marker of 123 bp.

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Table 2 Genotype frequencies of HPA-1, -3, and -5

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Genotypes

HPA-1

HPA-3

HPA-5

a/a a/b b/b

0.670 0.288 0.042

0.430 0.462 0.108

0.750 0.241 0.009

lanes. Similar PCR amplification patterns were obtained with primers related to the other plateletspecific alloantigens (HPA-3 and -5). Using this procedure, we typed 212 Southern Tunisian blood donors for HPA-1, -3, and -5 systems. Tables 2 and 3 summarize genotype and allele frequencies. There is no significant deviation from the Hardy–Weinberg equilibrium in any of the studied systems in this population (respectively, χ 2 0.55; 0.2 et 0.9). Table 3 showed also a comparison between our results and those observed in other studied populations.

Discussion The present study is the first to investigate allele frequency of the HPA-1, -3, and -5 systems in Southern Tunisian population and serves as an outline for future clinical research associated with platelet disorders in this group. A HPA-typed platelet of

Human platelets alloantigens polymorphisms

212 volunteers and regular blood donor registry was established. In our blood donors, a and b alleles frequencies of the three systems HPA-1, -3, and -5 are similar to those characterizing Caucasian populations. PCR-SSP, adapted in our laboratory, is the most used technique for HPA genotyping.7 It is a rapid, simple and non-laborious technique. PCR products are visualized directly after gel migration. The required reagents and equipments are standardized, which make PCR-SSP relatively inexpensive.13 In routine, the cost of HPA genotyping may be reduced without compromising the resolution. This is achieved by limiting the testing to the most involved systems in clinical pathology in a given population.14 For this reason, our choice is limited to three systems HPA-1, -3, and -5: the most involved among Caucasians.7 Genotyping results of southern Tunisia population are shown in Table 3. The comparison between our results and the literature showed that - HPA-1a allele frequency varies from north to south in Tunisia. In the centre, HPA-1a frequency was the lowest value observed in the literature (0.59 and 0.66). These frequencies are significantly lower than those found in our study.24,25 When compared to north Tunisia, our HPA-1a frequency showed no significant difference.23 Alleles distribution could be explained by predominant Berber influence in North

Table 3 Frequencies of HPA alleles in different ethnic groups Country

Ethnic groups

Chine16 Japon30

Chinese Blood donors Japanese Caucasian Major ethnic groups and castes of Pakistani population (Pathans and Sindhis) Caucasoid of mixed background Caucasian random platelet donors Random blood donor, estimated 99% Caucasian Caucasians from Northen Germany Caucasiens – Spanish Berber Blood donors (Sfax) Blood donors (Tunis) Blood donors (Tunis) Central Tunisia (Monastir) Sousse

Brazil20 Pakistan19

Austria21 France22 Germany18 Germany7 Croatia18 Algeria21 Spain18 Marocco15 Tunisia Tunisia23 Tunisia26,27 Tunisia24 Tunisia25

Effective

HPA-1a

HPA-1b

HPA-3a

HPA-3b

HPA-5a

HPA-5b

Method

1000 331

0.994* 0.991*

0.006† 0.009†

0.5945 0.718

0.4055 0.282

0.9860* 0.973*

0.014† 0.027†

PCR-SSP

100 593

0.925* 0.885*

0.075† 0.115†

0.600 0.69

0.400 0.31

0.920 0.9

0.080 0.1

PCR-RFLP PCR-SSP

911–906–931

0.852

0.148

0.612

0.388

0.892

0.108

PCR-RFLP

6192

0.848

0.152

0.62

0.38

0.874

0.126

PCR SSP

1583–1562–1643

0.84

0.16

0.60

0.40

0.92*

0.08†

PCR SSP

573

0.839

0.161

0.586

0.414

0.917*

0.083†

PCR SSP

430

0.17 0.173 0.19 0.252 0.186 0.25 0.27 0.335*

0.64 – 0.65 0.682 0.660 0.694 0.765

0.36 – 0.35 0.318 0.340 0.306 0.235

0.89 0.837 0.88 0.861 0.870 0.779† 0.665†

0.11 0.163 0.12 0.139 0.130 0.221* 0.335*

PCR-SSP

727–662–454 107–104–112 212 78–90–93 100 316

0.83 0.827 0.81 0.748 0.814 0.75 0.73 0.665†

444

0.59†

0.41*

0.75*

0.25†

PCR-SSP PCR-SSP PCR-RFLP PCR-SSP PCR-SSP PCR-SSP

RFLP, restriction-fragment-length polymorphism *Frequency significantly higher than Southern Tunisia. † Frequency significantly lower than Southern Tunisia.

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and South, consanguineous marriages and technical considerations. In fact, in our study, blood donors which are negative for HPA-1a allele, were systematically checked and sometimes proved positive, which was not reported in the methodology of other studies.23,26,27 - Compared to other countries, the prevalence of the HPA-1a allele among southern Tunisian (0.814) was lower than that reported for Asians (Chinese, Japanese, and Pakistani)16,19,29 and Brazilians.20 Nevertheless, the prevalence of this allele in south Tunisia was similar to those reported in other Caucasian populations.15,22 - In southern Tunisia, the HPA-3 polymorphism system shows a similar prevalence in comparison with all other populations so far studied. - When compared to northern Tunisia, HPA-5a frequency in our blood donors was significantly higher.23 Thus, the high frequency of 5b allele in north Tunisia could be explained by a large introduction of this allele through black slaves. The same conclusion was reported in studies on sickle cell anemia with the import of the Benin gene into Tunisia from black Africa.28 - HPA-5 allele frequencies analysis in southern Tunisia revealed a lower 5a allele frequency to those reported for Chinese16 and Japanese.29 No statistically significant difference was found when compared to Berbers of Morocco, French, and Brazilian Caucasians.15,20,22

These findings could be explained by the nature and the diversity of ethnic groups in Tunisian population. Indeed, Berbers, the original inhabitants of Tunisia and western part of North Africa, have been successively colonized and brewed with Phoenicians, Canaan (territory extended from Syria to Egypt through Lebanon and Palestine), Romans, Byzantines, Andalusians, Jews, Black slaves from trade between Sudan and Europe, and Turks.23

Conclusion The study of allelic polymorphism of HPA is very important, not only for anthropological and genetic reasons, but also to better predict the risk for alloimmunization for HPAs among distinct ethnic groups. The relative importance of each system HPA in anti-platelet allo-immunization varies among ethnic groups. Based on what has already been reported among Caucasians, our choice was limited to three systems HPA-1, -3, and -5.17 Allele frequencies of the three systems in south Tunisia were similar to those observed in Caucasians populations. The risk of immunization would join therefore that of Caucasians but seems to be less important than Northern Tunisia. This study allowed us to establish a HPA-typed platelet registry of 212 volunteers and regular blood

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donor. This registry could help in contacting blood donors of rare and negative phenotypes who are involved in platelets donation. The use of typed platelets is highly beneficial and even indispensable in certain diagnostic situations (research and identification of specific anti-platelet allo-antibodies) and/ or therapeutic (NAIT).

Acknowledgments This research was supported by CRTS (Centre Régional de Transfusion Sanguine de Sfax) and CBS (Centre de Biotechnologie de Sfax), Tunisia.

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