Two molecular polymorphisms to detect the (C)ce(s) type 1 haplotype. (2014)

Dear Sir,

Silvy and colleagues1 recently described a polymorphism restricted to the (C)ces type 1 haplotype, which allows this haplotype to be assayed by virtue of its 5′ breakpoint in RHD intron 3. (C)ces type 1 is a Rh variant, frequent in Africans and clinically relevant, for example in the treatment of sickle cell disease. The haplotype comprises an RHCE*ces allele of the RHCE gene linked to a hybrid RHD-CE-D allele of the RHD gene. In the hybrid, an RHCE-specific nucleotide sequence is embedded in an RHD-specific nucleotide sequence. This structural feature allows the hybrid to be defined by its two breakpoints, where the RHCE- and RHD-specific nucleotide sequences are fused.

The nucleotide sequence (accession n. {type:entrez-nucleotide,attrs:{text:HF536496,term_id:425702678,term_text:HF536496}}HF536496) and assay recently reported1 are in full accordance with a nucleotide sequence ({type:entrez-nucleotide,attrs:{text:AX135857,term_id:14272092,term_text:AX135857}}AX135857) which we identified and which has been online since 2001 (see Online Supplementary Appendix S1). Hence, this 5′ breakpoint does not commonly occur in exon 3, if at all, as has been published2,3, but rather in intron 3. We present here a complementary approach for the screening of (C)ces type 1 based on its second breakpoint, which is located in RHD intron 7 and represents the 3′ breakpoint (Table I): both breakpoints, 5′ and 3′ in the hybrid RHD-CE-D allele, are found in introns.



Table I

RHD intron segments for (C)ces type 1 and PCR primers.



During our research on the molecular basis of the prevalent RHD deletion4 and other RHD alleles expressing D negative or DEL phenotypes5, we established the 5′ (Online Supplementary Figure S1, encompassing the sequence of {type:entrez-nucleotide,attrs:{text:HF536496,term_id:425702678,term_text:HF536496}}HF536496) and 3′ breakpoint regions (Online Supplementary Figure S2) of a (C)ces haplotype (Online Supplementary Appendix S2), which was first encountered in VS antigen (RH20)-positive individuals of African origin6 and is known today as (C)ces type 13. We have designed a polymerase chain reaction assay with sequence-specific primers (PCR-SSP) to detect the 3′ breakpoint (Figure 1).



Figure 1

Gel electrophoresis of a PCR-SSP assay specific for (C)ces type 1.



To avoid the risk of transfusion deadlock in patients with sickle cell disease one or both assays for the (C) ces type 1 breakpoints may be used for clinical decision-making. Our assay may be more robust, because the primer Ccdes7b2 interrogates a polymorphism involving the deletion of several nucleotides in intron 7, a structural feature not available in intron 3 for assay design. While Silvy and colleagues showed that the 5′ breakpoint is specific for (C)ces type 11, the 3′ breakpoint of (C)ces type 2 remains unknown1,3. Our 3′ breakpoint assay is useful for detecting (C)ces type 1; additional study is required to confirm that our assay does not detect the uncommon (C)ces type 2.

There are at least 45 RHD-CE-D hybrid alleles known for the RHD gene, and the number of RHCE-D-CE hybrid alleles of the RHCE gene is expected to be as large. Hybrid alleles of both genes can occur in conjunction, forming a haplotype on one chromosome. They are caused by gene hybridisation, which is facilitated by the inverse orientation of the RHD and RHCE genes on the short arm of chromosome 15. Thus, detection of breakpoint regions may become a widely used feature in red cell genotyping and applications of dry matching, as exemplified by the current work.