Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and Comorian donors by

28 Pages·2017·0.58 MB·English

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￿
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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

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Journal: British Journal of Haematology

Manuscript ID: BJH-2011-00181.R1
P
Manuscript Type: Ordinary Papers
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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,
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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
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3 Running head: RHCE and KEL genotyping BJH-2011-00181(revised)
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7 Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and
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Comorian donors by multiplex SNaPshot and fragment assays: a transfusion support
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for sickle cell disease patients.
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Monique Silvy, Julie Di Cristofaro, Sophie Beley, Kassim Papa, Michel Rits, Pascale Richard
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Jacques Chiaroni and Pascal Bailly
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23 From the Laboratoire do’Hématologie Moléculaire, Établissement Français du Sang Alpes
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Méditerranée, UMR 6578, Université de la Méditerranée, Marseille, and the Établissement
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Français du Sang, Fort de France, Martinique, France.
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Corresponding author: Pascal Bailly, Laboratoire d’Hématologie Moléculaire, EFS Alpes
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35 Méditerranée, UMR 6578, Université de la Méditerranée, 207 Boulevard Sainte Marguerite,
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37 13009, Marseille, France; Phone: [phone]; FAX: [phone]; E-mail:
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[email protected]
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Address reprint requests to: Pascal Bailly, Laboratoire d’Hématologie Moléculaire, EFS Alpes
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44 Méditerranée, UMR 6578, Université de la Méditerranée, 207 Boulevard Sainte Marguerite,
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46 13009, Marseille, France; Phone: [phone]; FAX: [phone]; E-mail:
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[email protected]
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49

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