Complete RHD next-generation sequencing: establishment of reference RHD alleles

Abstract

The Rh blood group system (ISBT004) is the second most important blood group after ABO and is the most polymorphic one, with 55 antigens encoded by 2 genes, RHD and RHCE This research uses next-generation sequencing (NGS) to sequence the complete RHD gene by amplifying the whole gene using overlapping long-range polymerase chain reaction (LR-PCR) amplicons. The aim was to study different RHD alleles present in the population to establish reference RHD allele sequences by using the analysis of intronic single-nucleotide polymorphisms (SNPs) and their correlation to a specific Rh haplotype. Genomic DNA samples (n = 69) from blood donors of different serologically predicted genotypes including R₁R₁ (DCe/DCe), R₂R₂ (DcE/DcE), R₁R₂ (DCe/DcE), R₂R_Z (DcE/DCE), R₁r (DCe/dce), R₂r (DcE/dce), and R₀r (Dce/dce) were sequenced and data were then mapped to the human genome reference sequence hg38. We focused on the analysis of hemizygous samples, as these by definition will only have a single copy of RHD For the 69 samples sequenced, different exonic SNPs were detected that correlate with known variants. Multiple intronic SNPs were found in all samples: 21 intronic SNPs were present in all samples indicating their specificity to the RHD*DAU0 (RHD*10.00) haplotype which the hg38 reference sequence encodes. Twenty-three intronic SNPs were found to be R₂ haplotype specific, and 15 were linked to R₁, R₀, and R_Z haplotypes. In conclusion, intronic SNPs may represent a novel diagnostic approach to investigate known and novel variants of the RHD and RHCE genes, while being a useful approach to establish reference RHD allele sequences.

Graphical abstract

Figure 1.

The RHD and RHAG genes amplified in overlapping LR-PCR amplicons. (A) Six overlapping RHD LR-PCR amplicons. (B) Three overlapping RHAG LR-PCR amplicons.