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The RHD*weak D type 4.0 allele is predominantly but not exclusively cis‐associated with the altered RHCE*ce(c.48C, c.105T, c.733G, c.744C, c.1025T) allele in the French population

Dear Sir, In a recent paper published in Transfusion Medicine, Ouchari et al. (2013) reported a molecular study on the structure of the RHCE gene in cis with the weak D type 4.0 allele. This latter allele is characterised by the missense c.602C>G and c.667T>G variants resulting in the p.T201R and p.F223V amino acid substitutions, respectively, as well as the silent c.819G>A variant (Wagner et al., 1999). In their paper, the authors showed in 4 homozygous and 15 heterozygous Tunisian individuals that the aforementioned variant D allele is cis-associated with an altered RHCE*ce allele, which involves five single-base substitutions (i.e. c.48C and c.105T in exon 1, c.733G and c.744C in exon 5; and c.1025T in exon 7) already reported in other haplotypes but not combined together. In an effort to characterise the haplotype of the weak D type 4.0 allele carriers in our population of interest, and to check whether other RHCE alleles segregate with this allele, as already described (Hipsky et al., 2011; Reid et al., 2013), we investigated the RHCE gene in donors carrying this variant D genotype in our laboratory. Sixty-nine different, consecutive samples referred to our laboratory by the local sites of the French Blood Center (Etablissement Français du Sang, France) from November 2009 to November 2012 and genotyped as weak D type 4.0 by direct sequencing (Le Maréchal et al., 2007; Fichou et al., 2013a) were selected for the study. The exons of the RHCE gene were specifically PCR amplified with a combination of home-designed and previously published primers (Wagner et al., 2003) (Table S1). RHCE exons were individually amplified in a reaction mixture containing 1X HotStarTaq Master Mix (Qiagen, Courtaboeuf, France), 0·5 μM of both forward and reverse primers, and∼50 ng of template genomic DNA. PCR conditions were as follows: activation at 95 ◦C for 15 min; followed by 40 cycles of denaturation at 95 ◦C for 30 s, annealing at 60 ◦C for 30 s and extension at 72 ◦C for 30 s (except 2 min for exons 2 and 4); and a final extension step at 72 ◦C for 10 min. After treatment with ExoSAP-IT (USB, purchased from Ozyme, St Quentin en Yvelines, France), the purified PCR products served as templates for sequencing reactions using the BigDye Terminator v1.1 Cycle Sequencing Kit (Life Technologies, Saint Aubin, France) with each individual sequencing primer (Wagner et al., 2003). Products were directly sequenced in the following conditions: 96 ◦C for 1 min, followed