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 R(1)R(1) (DCe/DCe), R(2)R(2) (DcE/DcE), R(1)R(2) (DCe/DcE), R(2)R(Z) (DcE/DCE), R(1)r (DCe/dce), R(2)r (DcE/dce), and R(0)r (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 R(2) haplotype specific, and 15 were linked to R(1), R(0), 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.