Identification of novel polymorphism restricted to the <i>(C)ce</i><sup><i>s</i></sup><i>type 1</i> haplotype avoids risk of transfusion deadlock in <scp>SCD</scp> patients (2012)

The (C)ces type 1 haplotype is an Rh variant occurring in 7–15% of black individuals (Singleton et al, 2000). Molecular analysis of this haplotype has demonstrated a combination of two altered genes involving a hybrid RHD-CE-D gene segregated by a RHCE*ces allele of the RHCE gene. The hybrid RHD-CE-D gene comprises exons 1, 2, and part of exons 3, 8, 9 and 10 from RHD and encodes five nucleotide substitutions, i.e. 186G>T, 410C>T, and 455A>C in RHD exons and 733C>G and 1006G>T in RHCE exons (Faas et al, 1997). The RHCE*ces allele is a RHCE*ce allele encoding nucleotide substitutions at positions 733C>G and 1006G>T. The (C)ces type 1 variant presents a complex antigen profile with weak partial C and e, partial c, VS (RH20), and Rh42 that poses a special challenge for blood transfusion (Faas et al, 1997; Pham et al, 2009a). As (C)ces type 1 does not produce the high-prevalence antigens hrB (RH:-31) and HrB (RH:-34), it induces, in the homozygous state, the rare RH:-34 phenotype with a risk of transfusion deadlock. To date, the (C)ces type 1 haplotype has generally gone undetected until immunization occurs. Commercial monoclonal reagents are not able to detect altered C; red blood cells (RBCs) type C+ in most cases and routine serological tests do not identify altered RHCE alleles. The advent of molecular immunohaematology has provided a powerful adjunct to serological testing, thus improving selection of red cells for transfusion (Flickinger, 2006). However, an accurate genomic molecular method for detection of the (C)ces type 1 is still unavailable. The main problem for development of such a test is that none of the nucleotide substitutions currently identified are specific to the haplotype. To avoid the problems associated with using immunohaematological tools to study antigenic profile encoded by the (C)ces type 1 haplotype, we undertook investigations to find a polymorphism(s) specific to (C)ces type 1, allowing its detection by molecular analysis. For this purpose we sequenced the RHD introns in the vicinity of exons (Table S1). In addition, we studied a zone of the 5′UTR of the (C)ces type 1 haplotype in which a small DNA stretch of at least 13 bp has been replaced by the corresponding RHCE sequence (Wagner et al, 2001). Comparison of intron sequences in five DNA samples bearing a (C)ces type 1 haplotype with 11 wild type RHD alleles consistently revealed a shared pattern composed of 24 polymorphisms and two STRs distributed between the promoter and intron 3 (Table 1). To determine the specificity of these polymorphisms, we analysed 45 samples bearing alleles from the DIVa (n = 14), weak D type 4 (n = 17) and DAU (n = 14) African clusters. A single nucleotide polymorphism (SNP: IVS5-16c>t) restricted to samples with RHD*weak D type 4.0 or RHD*DIIIa alleles was identified, confirming the hypothesis that RHD*DIIIa results from recombination of the RHD*weak D type 4.0 and RHD*DIVa alleles (Wagner et al, 2002; Pham et al, 2009a). The RHD*DAR and (C)ces type 1 had a total of 8 SNPs in common. These findings are consistent with the hypothesis of gene conversion inferred from the variation profile showing a number of repetitive patches. It is noteworthy that the RHD*DIVa2 and RHD*DIIIa alleles shared all the polymorphisms observed in (C)ces type 1 haplotype, including the 13 bp insertion in the promoter region. Interestingly, despite the homogenous profile of (C)ces type 1 haplotypes, both RHD*DIVa2 and RHD*DIIIa showed three different intron patterns. None of the 24 polymorphisms identified was specific to the (C)ces type 1 haplotype. In contrast to initial description we observed that both exon 3 and the upstream part of intron 3 originated from the RHD gene with typical features of the DIVa cluster (410C>T, 455A>C, IVS3+9t>c, IVS3+41g>a, IVS3+69a>g, IVS3+153g>a, IVS3+165c>t). This observation strongly suggested that the RHD/CE breakpoint was located in intron 3. Sequencing of overlapping regions of intron 3, starting from exon 3, located RHD/RHCE breakpoint between 3·0 and 3·2 kb downstream exon 3. A primer pair allowing specific polymerase chain reaction amplification of hybrid gene was blindly tested on 118 samples (see Appendix S1). All samples with a documented (C)ces type 1 haplotype (n = 52) showed amplification while all other samples were negative regardless of the RHD and RHCE alleles present. Samples with (C)ces type 2 haplotype, which results from a RHCE*ces conversion into a conventional RHD allele segregated with a RHCE*ces allele, were also negative (n = 4) (Pham et al, 2009a). These findings strongly indicated that the breakpoint is a uniquely specific feature of the (C)ces type 1 haplotype. Sequencing of (C)ces type 1-specific amplicon revealed the IVS3+3100a>g SNP in all samples (Fig 1). The specificity of this SNP was subsequently confirmed by sequencing the breakpoint region of RHD in 66 samples expressing either wild type RHD, or partial/weak phenotype and in four samples with the (C)ces type 2 haplotype. The precise location of the RHD/RHCE breakpoint in intron 3 of the RHD gene and identification of the IVS3+3100a>g SNP specific to (C)ces type 1 open new possibilities for the development of molecular detection tests in both patients and donors. This is of major importance in transfusion medicine because this haplotype is commonly found in patients with sickle cell disease (SCD) who have developed complex Rh antibodies including anti-HrB and -hrB and need transfusion of HrB- and hrB- RBCs (Chou & Westhoff, 2011). Given that different molecular backgrounds are associated with lack of HrB and hrB antigens, not all phenotypically similar hrB- units will be compatible (Pham et al, 2009a,b). For this reason, matching the Rh genotype of the donor and patient is important to prevent further Rh sensitization. To locate hrB– donors for patients with SCD, molecular analysis should be implemented along with the screening programme proposed by Gammon and Velasquez (2002). High-throughput genotyping platforms have been shown to significantly improve success of searches for rare types blood (LaSalle-Williams et al, 2011). The findings of this study will be medically valuable not only to allow accurate and specific detection of the (C)ces type 1 haplotype in patients but also to facilitate identification of suitable rare blood donors to avoid allo-immunization, especially in the context of SCD requiring chronic transfusion therapy with high risk of immunization. We wish to thank Thierry Peyrard (CNRGS-INSERM U665, Paris) and Christophe Tournamille (Etablissement Français du Sang Créteil) for providing the (C)ces type 2 gDNA samples used in this study. MS developed the assays, analysed the data and wrote the paper, TG and SB performed the analysis, JC assisted in study design and PB designed the research, analysed the data and wrote the paper. MS, TG, SB, JC and PB are all employees of Établissement Français du Sang and all authors declare they have no conflict of interest in the subject matter of their article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.