Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and Comorian donors by multiplex SNaPshot and fragment assays: a transfusion support for sickle cell disease patients Monique Silvy, Julie Di Cristofaro, Sophie Beley, Kassim Papa, Michel Rits, Pascale Richard, Chiaroni Jacques, Pascal Bailly To cite this version: Monique Silvy, Julie Di Cristofaro, Sophie Beley, Kassim Papa, Michel Rits, et al.. Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and Comorian donors by multiplex SNaPshot and fragment assays: a transfusion support for sickle cell disease patients. British Journal of Haematology, 2011, 154 (2), pp.260. ￿10.1111/j.1365-2141.2011.08691.x￿. ￿hal-00645380￿ HAL Id: hal-00645380 https://hal.science/hal-00645380 Submitted on 28 Nov 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. British Journal of Haematology Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and Comorian donors by multiplex SNaPshot and fragment assays: a transfusion support for sickle cell F disease patients o r Journal: British Journal of Haematology Manuscript ID: BJH-2011-00181.R1 P Manuscript Type: Ordinary Papers e Date Submitted by the 17-Mar-2011 Author: e Complete List of Authors: SILVY, Moniqrue; Etablissement Français du Sang Alpes- Méditerranée, La b. d'Hématologie Moléculaire, UMR6578, Uni. de la Méditerranée DI CRISTOFARO, Julie; Etablissement Français du Sang Alpes- R Méditerranée, Lab. d'Hématologie Moléculaire, UMR6578, Uni. de la Méditerranée e BELEY, Sophie; Etablissement Français du Sang, Lab. d'Hématologie Moléculaire, UMR6578, Uni. de la Méditerranée v PAPA, Kassim; Etablissement Français du Sang Alpes-Méditerranée, i Lab. d'Hématologie Moléculaire, UMR6578, Uni. de la Méditerranée RITS, Michel; Etablissement Français du Sang, Martinique e RICHARD, Pascale; Etablissement Français du Sang, Martinique Jacques, CHIARONI; Etablissement Français du Sang Alpes- Méditerranée, Lab. d'Hématologie Moléculaire, UMR6578, Uni. de la w Méditerranée BAILLY, Pascal; Etablissement Français du Sang Alpes- Méditerranée, Lab. d'Hématologie Moléculaire, UMR6578, Uni. de la Méditerranée BLOOD TRANSFUSION, MOLECULAR BIOLOGY, RED CELL Key Words: ANTIGENS, SICKLE CELL DISEASE Page 1 of 26 British Journal of Haematology 1 2 3 Running head: RHCE and KEL genotyping BJH-2011-00181(revised) 4 5 6 7 Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and 8 Comorian donors by multiplex SNaPshot and fragment assays: a transfusion support 9 10 for sickle cell disease patients. 11 12 13 14 15 Monique Silvy, Julie Di Cristofaro, Sophie Beley, Kassim Papa, Michel Rits, Pascale Richard 16 17 Jacques Chiaroni and Pascal Bailly 18 19 20 F 21 22 23 From the Laboratoire do’Hématologie Moléculaire, Établissement Français du Sang Alpes 24 r Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, and the Établissement 25 26 Français du Sang, Fort de France, Martinique, France. 27 P 28 29 e 30 31 e 32 r 33 Corresponding author: Pascal Bailly, Laboratoire d’Hématologie Moléculaire, EFS Alpes 34 35 Méditerranée, UMR 6578, Université de la Méditerranée, 207 Boulevard Sainte Marguerite, R 36 37 13009, Marseille, France; Phone: 33-1-4-91-17-01-74; FAX: 33-1-4-91-17-01-78; E-mail: e 38 [email protected] 39 v 40 i 41 42 e Address reprint requests to: Pascal Bailly, Laboratoire d’Hématologie Moléculaire, EFS Alpes 43 44 Méditerranée, UMR 6578, Université de la Méditerranée, 207 Boulevard Sainte Marguerite, w 45 46 13009, Marseille, France; Phone: 33-1-4-91-17-01-74; FAX: 33-1-4-91-17-01-78; E-mail: 47 [email protected] 48 49 50 51 This study was supported by Établissement Français du Sang, Paris, France (all co-authors). 52 53 54 55 56 57 58 59 60 The authors declare that they have no conflict of interest in the subject matter of their manuscript. British Journal of Haematology Page 2 of 26 1 2 3 ABSTRACT 4 5 6 7 To lower the alloimmunization risk following transfusion in blacks, we developed two 8 9 genotyping assays for large-scale screening of Comorian and Afro-Caribbean donors. One 10 was a multiplex SNaPshot assay designed to identify ces(340), ceMO/AR/EK/BI/SM, ces, 11 12 ces(1006) and KEL*6/*7 alleles. The other was a multiplex fragment assay designed to detect 13 14 RHD, RHDψ and RHCE*C and 455A>C transversion consistent with (C)ces Type 1 and DIII 15 16 Type5 ces. 17 18 Variant RHCE*ce alleles or RH haplotypes were detected in 58.69% of Comorians 19 and 41.23% of Afro-Caribbeans. The ces allele, (C)ces Type 1, and DIII Type 5 ces haplotypes 20 F 21 were identified respectively in 39.13%, 14.67% and 4.88% of Comorians and 32.23%, 5.28% 22 o 23 and 1.76% of Afro-Caribbeans. Genotypes consistent with partial D, C, c and/or e antigen 24 r 25 expression were observed in 26.08% of Comorians and 14.69% of Afro-Caribbeans. No 26 homozygous genotype corresponding to the RH:-18, -34, and -46 phenotypes were found. 27 P 28 However, over 50% of genotypes produced low-prevalence antigens at risk for negative 29 e 30 recipients, i.e., V, VS, JAL, and/or KEL6. One new variant RHCE*ces(712) allele was 31 e 32 identified. r 33 34 This is the first determination of variant RHCE and KEL allele frequencies. Results indicate 35 most suitable targets for molecular assay screRening to optimize use of compatible blood units 36 37 and lower immunization risk. e 38 39 v 40 i 41 42 e 43 44 w 45 46 47 48 49 50 51 52 Abstract = 200 words 53 54 KEY WORDS: RHCE and KEL gene variants, SNaPshot, genotype, population with African 55 56 origin. 57 58 ABBREVIATIONS: SNP(s) = single nucleotide polymorphism (s); bp = base pairs; rfu(s) = 59 60 relative fluorescence unit(s). Page 3 of 26 British Journal of Haematology 1 2 3 INTRODUCTION 4 5 The risk of post-transfusion alloimmunization increases with donor exposure and 6 7 degree of donor/recipient red blood cell (RBC) antigen disparity (Higgins & Sloan 2008). The 8 9 risk is particularly high for minority racial groups such as persons of African descent living in 10 Europe who harbor molecular variants compared to European blood donors (Aygun, et al, 11 12 2002; Daniels 2002; Noizat-Pirenne 2003). Sickle cell disease (SCD) patients undergoing 13 14 long-term transfusion therapy have a higher risk of alloimmunization than the general 15 16 population: 20 to 40 % versus 5% (Flickinger 2006). For SCD patients, alloimmunization has 17 severe clinical consequences not only by increasing the difficulty of finding compatible blood 18 19 but also by causing hemolytic transfusion reaction and autoantibody production and reducing 20 F 21 the longevity of transfused RBCs (Aygun et al, 2002). Consistent with the fact that blacks 22 o 23 harbor more molecular Rh variants than European blood donors (Avent & Reid 2000), it has 24 r 25 been observed that more than 60% of alloantibodies in SCD patients are directed against Rh 26 antigens (Chou & Westhoff 2009; Isaak, et al, 2006). 27 P 28 Two strategies that have been implemented in several countries to lower the incidence 29 e 30 of alloantibody production in SCD patients, are extended Rh and Kell phenotype matching 31 e 32 and intra-ethnic blood transfusions (Soslerr et al, 1993; Wayne et al, 1993). Although these 33 approaches seem to have positive effects to reduce alloimmunization (Vichinsky et al, 2001; 34 35 Tahhan et al, 1994), it appears that some SCRD patients still produce anti-D, -C , -c and –e 36 37 despite having type D+, C+, c+ and e+ RBCs resepectively, that may be incorrectly classified 38 39 as auto antibodies (Westhoff 2008). Indeed, molecuvlar analysis of RHD, RHCE, KEL and 40 i 41 MNS genes in SCD patients has identified nucleotide polymorphisms showing that Rh, Kel 42 e and Glycophorin B polypeptides differ from conventional amino-acid sequences indicating 43 44 that serum antibodies are allo-type. w 45 46 Three types of variant antigens are encountered : (i) absence of high-prevalence 47 48 antigens resulting from homozygous alleles, haplotypes or gene deletion, (ii) partial antigens 49 (c, e, C, D, Uvar), and (iii) expression of low-prevalence antigens (see Table 1). These 50 51 phenotypes are generally recognized once the allo-immunization is elicited against missing 52 53 epitopes or when a transfusion reaction has occurred. 54 55 These antigen variants hardly recognized by current serological reagents, can be well 56 57 identified by molecular tools. Despite this advance and different genotype reports on Afro- 58 Caribbean and African American black individuals notably with sickle cell disease, guidelines 59 60 on the frequencies of variant RHCE alleles and KEL*6/*7 alleles from large cohorts of British Journal of Haematology Page 4 of 26 1 2 3 African origin would be useful to prevent alloimmunization and improve transfusion therapy 4 5 in countries with large African immigrant populations. 6 7 Our transfusion center is located in the southern French department of Bouches-du- 8 Rhône that has a large Comorian and Afro-Caribbean community (Chiaroni et al, 2004). The 9 10 presence of this large minority population poses a major transfusional challenge because of 11 12 the high frequency of blood diseases such as SCD. To allow routine evaluation of 13 14 alloimmunization risk, we have developed two simple and clinically useful genotyping assays 15 based on the well-known SNaPshot method (Di Cristofaro et al, 2010; Silvy et al, 2011). One 16 17 is a multiplex primer extension assay (SNaPshot assay) allowing analysis of nucleotides at 18 19 key positions 340, 667, 712, 733 and 1006 of the RHCE gene and position 1910 of the KEL 20 21 gene designed to ideFntify ces(340), ceMO/AR/EK/BI/SM, ces, ces(1006) and KEL*6/*7 alleles. 22 23 The other is a multipleox fragment assay allowing the detection of the presence of RHD, 24 r RHDψ and RHCE*C genes as well as (C)ces Type 1 and DIII Type 5 ces haplotypes (Pham et 25 26 al, 2009b; Poulter et al, 1996; Singleton et al, 2000). 27 P 28 The purpose of this report is to describe the results of a prospective study carried out 29 e 30 after validation of the specificity and reliability of these two molecular assays. Study was 31 e carried out on two cohorts of African descent, i.e., 184 Comorians living in the south of 32 r 33 France and 1021 Afro-Caribbean donors from Martinique Island. Our goal was to determine 34 35 their RHCE and KEL polymorphisms and to hRave a better knowledge of the allelic frequencies 36 37 to minimize production of Rh and Kell allo antibodies, to reduce complications and improve e 38 39 transfusion therapy. v 40 i 41 42 e 43 44 w 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 5 of 26 British Journal of Haematology 1 2 3 MATERIAL AND METHODS 4 5 Blood samples and immunohematology 6 7 Blood samples were collected on ethylenediaminetetraacetate tubes from 184 Comorian 8 9 immigrants from Grande Comore Island (Njazidja) living in Marseille, France and from 1021 10 unrelated Afro-Caribbeans living on Martinique Island. The main selection criteria for 11 12 sampling was donor origin from the Comoros or Martinique Island, i.e., place of birth mother, 13 14 father and all four grandparents. The study protocol was approved by the Comité de 15 16 Protection des Personnes dans la Recherche Biologique (CPPRB) in France. 17 Red cell serological testing was carried out at the Établissement Français du Sang (EFS) 18 19 facilities in Marseille and Fort de France using commercially available MoAb reagents. 20 F 21 Diagast reagents (Loos, France) were used to test the following specificities: anti-RhD (RH1, 22 o 23 clones: P3x61 + P3x21223B10 + P3x290 + P3x35), anti-RhC (RH2, clones: P3x25513G8 + 24 r 25 MS24), anti-RhE (RH3, clone : 906), anti-Rhc (RH4, clone: 951), and anti-Rhe (RH5, clones: 26 P3GD512 + MS63). Eurobio reagents (Les Ulis, France) were used to test anti-RhD (RH1, 27 P 28 clone: CAZ), anti-RhC (RH2, clone: MS24), anti-RhE (RH3, clone: MS35), anti-Rhc (RH4, 29 e 30 clone: 906) and anti-Rhe (RH5, clones: MS16 + MS21 + MS63). Combinations of several 31 e 32 anti-RhD reagents reacting with the morst common partial RhD antigens in the European 33 population particularly RhDVI were also used. Standard serological tests were performed 34 35 according to the manufacturer’s instructioRns, but indirect antiglobulin tests were not 36 37 performed to detect some weak RhD variants. e 38 39 v 40 i 41 Genomic DNA extraction 42 e 43 Genomic DNA was extracted from 200 µl of whole blood using the MagNA Pure LC DNA 44 w 45 Isolation Kit (Roche, Meylan, France) according to the manufacturer’s instructions. DNA was 46 eluted in 100 µl water and quantified by measuring optical density. 47 48 49 50 Detection of RHCE and KEL polymorphisms by multiplex PCR, primer extension assay, and 51 52 capillary electrophoresis analysis 53 A multiplex PCR was developed to amplify RHCE-gene exons 3, 5 and 7 and exon 17 of KEL 54 55 gene (Table 2). Amplification was performed using a PTC200 thermocycler (Biorad, France) 56 57 on 200 ng of genomic DNA in a final volume of 25 µl containing PCR Qiagen Master Mix, Q 58 59 solution (Qiagen, Courtaboeuf, France), and primer sets (see Table 2 for final concentration). 60 After an initial denaturation step at 95°C for 15 minutes, a total of 35 cycles were carried out British Journal of Haematology Page 6 of 26 1 2 3 using the following sequence: denaturation at 95°C for 30 seconds, primer annealing at 57°C 4 5 for 90 seconds, and polymerization at 72°C for 90 seconds. Amplification was terminated 6 7 after a 10 min-extension at 72°C. Amplicons were controlled on 2% (wt/vol) agarose gel. 8 Purification of amplified products (10 µl) were performed using 10 units of Exonuclease I and 9 10 3.3 units of shrimp alkaline phosphatase (Euromedex, Souffelweyersheim, France) for 60 11 12 minutes at 37°C followed by enzyme inactivation for 15 minutes at 80°C. The next step 13 14 consisted of single-base primer extension using the SNaPshot kit (Applied Biosystems, 15 Courtaboeuf, France) according to the manufacturer’s protocol. This reaction was carried out 16 17 in a final volume of 10 µl containing 3 µl of purified PCR products, 5 µl of SNaPshot ready 18 19 reaction premix, and extension primers (see Table 2 for final concentration). The extension 20 F 21 reaction included 25 cycles of denaturation at 96°C for 10 seconds, annealing at 50°C for 5 22 23 seconds, and extensiono at 60°C for 30 seconds. After treatment with shrimp alkaline 24 r phosphatase, 0.5 µl of the SNaPshot primer extension reaction product was mixed with 25 26 formamide and 120 LIZ ladder before analysis on a capillary sequencer (ABI PRISM3130XL, 27 P 28 Applied Biosystems) using POP 7 polymer according to the manufacturer’s instructions. Data 29 e 30 were analyzed using GeneMapper v4.0 software (Applied Biosystems). For each SNaPshot 31 e analysis, positive and negative controls were performed by replacing genomic DNA with 32 r 33 already genotyped DNA and H O (DNase/RNase free, Invitrogen) respectively. 34 2 35 R 36 37 Multiplex PCR and capillary electrophoresis analysis e 38 39 A multiplex fragment assay was developed to devtect the presence of RHD, RHDψ and 40 i RHCE*C genes and to reveal 455A>C transversion located in RHD exon-3 consistent with 41 42 (C)ces Type 1 and DIII Type 5 ces haplotypes. Amplificatieon of the CCR5 exon-2 was used as 43 44 an internal control. Amplification was performed using a PwTC 200 thermocycler (Biorad, 45 46 France) on 200 ng of genomic DNA in a final volume of 25 µl containing PCR buffer, 47 3 mmol/l MgCl , 0.2 mmol/l of each dNTP, 1 unit of Taq DNA polymerase (Invitrogen, 48 2 49 Cergy Pontoise, France), and primers (see Table 3 for final concentrations). Conditions for 50 51 multiplex PCR were as follows: 95°C for 5 minutes followed by 40 cycles of 95°C for 30 52 53 seconds, 59°C for 45 seconds, 72°C for 2 minutes, and 72°C for 7 minutes. As in the previous 54 55 test, 1.5 µl of PCR reaction product was mixed with formamide and 500 LIZ ladder before 56 analysis on a capillary sequencer (ABI PRISM3130XL, Applied Biosystems) using software 57 58 (GeneMapper v4.0, Applied Biosystems). In each experiment, positive and negative controls 59 60 were performed with already genotyped DNA and H O (DNase/RNase free, Invitrogen) 2 respectively. Page 7 of 26 British Journal of Haematology 1 2 3 4 5 Further gene analysis 6 7 To distinguish between partial DIII Type 5 ces and (C)ces Type 1 consistent with a 455A>C 8 transversion located in RHD exon-3 revealed by multiplex fragment assay and to identify 9 10 DAR linked to ceAR and ceEK alleles, the previously reported SNaPshot assay (Silvy et al, 11 12 2011) for detection of weak D and DEL alleles was used to analyze RHD positions 602, 667, 13 14 819 and 1025. To distinguish between ceEK, ceBI and ceSM alleles, revealed by the 712A>G 15 transition on SNaPshot assay, and between ceEK, ceBI or ceSM in trans to a ces allele and 16 17 ceAR, revealed by 712A>G and 733C>G nucleotide changes, RHCE exon-5 were amplified 18 19 by PCR from genomic DNA using sense primer (5’-gcaacagagcaagagtcca-3’, nt –310 to –292) 20 F 21 and anti-sense primer (5’-actccccccagaaagcctttg-3’, nt +222 to +202). PCR products were 22 23 then cloned into pGEMo-T vector (Promega) and sequenced using a sequencing kit (BigDye 24 r Terminator v1.1, Applied Biosystems) and a genetic analyzer (ABI PRISM3130XL, Applied 25 26 Biosystems) according to manufacturer’s protocol. To distinguish between ceBI and ceSM 27 P 28 alleles, RHCE exon-8 was amplified and sequenced to determine the nucleotide at position 29 e 30 1132. RN haplotype from samples typed DCCee was investigated to identify the RH:-46, 31 e RH32 phenotype, using TaqMan assay as previously described (Tournamille et al, 2010). 32 r 33 34 35 R 36 37 e 38 39 v 40 i 41 42 e 43 44 w 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 British Journal of Haematology Page 8 of 26 1 2 3 RESULTS 4 5 Validation of assays 6 7 The first assay, i.e., multiplex PCR coupled with a single base extension reaction using 8 genomic DNA, was designed for simultaneous determination of 6 SNPs defining 6 variant 9 10 RHCE*ce alleles and KEL*6/*7 alleles observed in individuals of African descent. The 11 12 primer sets selected to amplify RHCE exons 3, 5, 7 and KEL exon 17 are shown in table 2. 13 14 For each PCR, the specificity of amplicons was evaluated in singleplex and multiplex 15 reactions in which PCR conditions, DNA template, and primer concentrations were adjusted 16 17 to obtain bands of similar intensity (data not shown). The entire assay including multiplex 18 19 PCR, primer extension (see Table 2), electrophoresis, and fluorescent typing step was 20 F 21 calibrated to obtain electrophoretic peaks of at least 500 rfus at regular intervals. Genomic 22 23 DNA samples already ogenotyped for RHCE*ce variants and/or KEL*6/*7 were used to 24 r validate the SNaPshot signal at each assay position, i.e., 340, 667, 712, 733 and 1006 of the 25 26 RHCE and 1910 of the KEL gene, The validation set included ces (n=8, four homozygous 27 P 28 samples), (C)ces Type 1 (n=10, one homozygous sample), DIII Type 5 ces (n=4, one 29 e 30 homozygous sample), ces(340) (n=1, heterozygous form), ceAR (n=6, all in trans to a ce 31 e allele), ceEK (n=1, heterozygous form), and ceMO (n=10, one homozygous sample). Also 32 r 33 included in the validation set were ten KEL*7/*7, five KEL*6/*7, and three KEL*6/*6 34 35 samples. R 36 37 The second genomic DNA assay, i.e., multiplex PCR using 8 primer sets each e 38 including one labeled primer (see Table 3), was designed for simultaneous amplification of 39 v 40 wild type RHD exon-3, -4, -5 and -10, RHD exon-3 (i455A>C), RHDΨ exon-4, RHCE*C 41 42 e intron-2, and CCR5 exon-2 as positive control. After optimization of singleplex and multiplex 43 44 reaction conditions as described above, multiplex fragment signals were validated using a w 45 46 validation set composed of genomic DNA samples genotyped RHDΨ (n = 6, one of them with 47 RHCE*C allele), (C)ces Type 1 (n = 10, one with RHCE*C allele), RHD deletion (del/del, n = 48 49 6, four of them with RHCE*C allele), and RHD (n=10, five with RHCE*C allele). 50 51 Results obtained with both assays were fully concordant with known RHDCE and 52 53 KEL genotypes. Typical GeneMapper electropherograms obtained after capillary 54 electrophoresis in a POP7 polymer system are presented in figure 1 showing the relevance of 55 56 the two molecular analyses at polymorphic positions 340C>T, 667G>T, 712A>G, 733C>G 57 58 and 1006G>T of the RHCE gene and 1910G>T of the KEL genes (panel A) and wild type 59 60 RHD exon-3, -4, -5 and -10, RHD exon-3 (455A>C), RHDΨ exon-4, RHCE*C intron-2 and CCR5 exon-2 as positive control (panel B). Electrophoregram interpretations are given in