Towards haplotypes of blood group genes: the impact of long-read sequencing in molecular immunohematology

Among the currently recognized 48 human blood group systems with more than 370 blood group antigens, the Rh system, defined by the two homologous genes RHD and RHCE, is the most polymorphic. Both genes are closely located on chromosome 1 separated by only 32 kilo bases (kb) (Figure 1). By January 2026, the International Society of Blood Transfusion (ISBT) Blood Group Database listed 447 RHD alleles and 186 RHCE alleles including the reference alleles (1). Most alleles are defined by single nucleotide variations (SNVs). In addition, gene conversion events generated a variety of RHD-RHCE hybrid alleles due to the high sequence homology (over 93%) of the two genes. The Rh system comprised 56 antigens, including the five major antigens D, C, c, E and e. When the serologic phenotype is unclear (weak or partial antigen), the investigation of the molecular background is usually conducted by using specific polymerase chain reaction (PCR) methods for SNV genotyping or by short-read sequencing RHD and RHCE. The correct assignment of variants to one or the other allele can impact the interpretation of genotype-phenotype correlation. However, haplotype phasing of variants is often not possible by specific PCR methods or short-read sequencing.

Figure 1 RH gene locus on chromosome 1 of the human genome GRCh38 spanning approximately 150 kb. The RHD and RHCE genes each have 10 exons and are oriented in the opposite direction. The non-homologous TMEM50A gene is located in between. Created in BioRender. Wörner L. [2026]. https://BioRender.com/limlf99.

With the introduction of long-read sequencing technologies such as single molecule real-time (SMRT) sequencing [Pacific Biosciences (PacBio)] and nanopore sequencing [Oxford Nanopore Technologies (ONT)] the characterization of gene haplotypes became more feasible. These technologies have a significant impact on blood group genetics and facilitate the setup of reference haplotype data for all blood group genes as several studies have already shown. For instance, long-read sequencing of the ABO gene, coding for the ABO blood group system, enabled the definition of haplotypes and a phylogenetic analysis (2). The ACKR1 gene for the Duffy blood group system was analyzed by ONT sequencing of a single 5.7 kb PCR product and 25 alleles were defined by phasing of the identified SNVs in a different research work (3). The MNS blood group system is defined by the homologous GYPA, GYPB and GYPE genes. SMRT sequencing of large amplicons yielded standardized reference haplotype sequences, which can be used for MNS genotyping and identification of hybrid genes (4). As another example for the investigation of the RH locus, Zang et al. (5) used targeted DNA capture, SMRT long-read sequencing, and bioinformatics to fully assemble the RH region. Consensus sequences of 2.1 to 2.9 kb average length enabled phasing of distant SNVs to determine RHD-RHCE haplotypes, and identification of known and novel structural variations along with the breakpoints.

Recently, Song et al. (6) described the application of SMRT high-fidelity long-read sequencing to the entire RH gene locus by using overlapping amplicons. For the targeted so-called “meticulous amplicon tiling” protocol, 32 long-range polymerase chain reaction (LR-PCR) amplicons with a length of 25 kb each are combined. This allowed the generation of 170 kb long full-length RHD-RHCE haplotype sequences, spanning RHD, the intergenic region and RHCE. With the analysis of 63 samples, the setup of a full-length RHD-RHCE reference haplotype collection of the Eurasian population was aimed. For all samples, serological phenotypes matched the identified haplotypes. Complete RHD-RHCE phasing was achieved for 48 samples, from which 96 allelic full-length reference haplotype sequences were derived. Partially phased haplotype sequences (either complete RHD or RHCE phasing) were used to create shorter reference haplotype sequences. The 96 allelic full-length reference haplotype sequences were phylogenetically analyzed. The authors also focused on the detection of structural variants, such as RHD zygosity, rhesus box cross-overs, or gene conversion events in either RHD or RHCE, and were able to identify a variety of variants with their respective breakpoints. In summary, the introduction of the long-read sequencing-based approach leveraged the potential of this technology to analyze the highly homologous RH gene locus, thereby providing new perspectives for the molecular basis of RH haplotypes and their evolutionary history.

The study of Song et al. (6) provides an important insight into current technological developments to expand the toolbox of molecular immunohematology. The straight-forward approach demonstrated the potential of long-read sequencing to overcome limitations of current methods, particularly for the analysis of homologous blood group genes. Sequencing of LR-PCR amplicons enables accurate phasing of variants covering long distances (7). The analysis of haplotype sequences improves the interpretation of the blood group phenotype, especially in the context of compound heterozygosity and novel variants (7). Long-read sequencing of blood group genes can also help to resolve phenotype-genotype discrepancies (7,8). In addition, the technique offers great potential for sequencing genetic regions with high sequence homology, such as the RH gene locus, but also GYPA and GYPB of the MNS blood group system. While homologous regions are difficult to amplify specifically in targeted sequencing approaches and short-read sequences often show mapping errors, long-read sequencing can overcome these limitations, as demonstrated by Song et al. (6). Prospectively, the “meticulous amplification tiling” protocol applied to the PacBio sequencing technology could also be transferred to other homologous (blood group) genes.

The study highlights another important aspect of long-read sequencing: the need for the systematic generation of high-quality haplotype reference sequences of genes and also of the entire human genome (9,10). Since the majority of the currently available blood group allele data are limited to single variants in exons, further research work is needed to characterize high-quality reference haplotype sequences. The setup of reference haplotype sequence data is essential to improve the reliability of bioinformatic-based variant calling and genotype-phenotype correlation. It is also highly important to make such haplotype reference sequences publicly available so that this data can be accessed regardless of available resources. However, the high costs of comprehensive and systematic research approaches have often hampered to generate haplotype sequences collections. High costs are also an important issue for a possible implementation in a in a clinical diagnostic setting. Besides the expectable reduction in costs and time due to further technical improvement, one could also think of long-read nanopore sequencing as a probably more cost-effective alternative technology.

Song et al. (6) presented a comprehensive long-read sequencing approach for the generation of haplotype sequences as a more comprehensive basis for sequence interpretation. Current challenges of sequence analysis and interpretation for homologous blood group genes were addressed as well. As admitted by the authors, the identified haplotype sequences do not represent the whole diversity and complexity of the RHD-RHCE region due to the restricted sample set. In addition, there are still challenges regarding the implementation in a routine diagnostic setting. However, the presented long-read sequencing approach can be used to improve reference data quality as an important step towards comprehensive reference sequence data collections for all blood group systems.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Translational Medicine. The article has undergone external peer review.

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-2026-1-0022/prf

Funding: None.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-2026-1-0022/coif). The authors have no conflicts of interest to declare.

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