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), R2RZ (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 RZ 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.
Digital PCR provides a highly accurate method to rapidly define blood group zygosity and has clinical application in the analysis of Rh phenotyped or genotyped samples. The vast majority of current blood group genotyping platforms are not designed to define zygosity, and thus, this technique may be used to define paternal zygosity in pregnancies that are at a risk of hemolytic disease of the fetus and newborn and can distinguish between homo- and hemizygous-positive individuals.
Fetal RHD screening for targeted routine antenatal anti-D prophylaxis has been implemented in many countries, including Finland, since the 2010s. Comprehensive knowledge of the RHD polymorphism in the population is essential for the performance and safety of the anti-D prophylaxis program. During the first 3 years of the national screening program in Finland, over 16 000 samples from RhD− women were screened for fetal RHD; among them, 79 samples (0.5%) containing a maternal variant allele were detected. Of the detected maternal variants, 35 cases remained inconclusive using the traditional genotyping methods and required further analysis by next-generation sequencing (NGS) of the whole RHD gene to uncover the variant allele. In addition to the 13 RHD variants that have been previously reported in different populations, 8 novel variants were also detected, indicating that there is more variation of RHD in the RhD− Finnish population than has been previously known. Three of the novel alleles were identified in multiple samples; thus, they are likely specific to the original Finnish population. National screening has thus provided new information about the diversity of RHD variants in the Finnish population. The results show that NGS is a powerful method for genotyping the highly polymorphic RHD gene compared with traditional methods that rely on the detection of specific nucleotides by polymerase chain reaction amplification.
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