Application of Blood Group Genotyping by Next-Generation Sequencing in Various Immunohaematology Cases

Abstract

Background: Next-generation sequencing (NGS) technology has been recently introduced into blood group genotyping; however, there are few studies using NGS-based blood group genotyping in real-world clinical settings. In this study, we applied NGS-based blood group genotyping into various immunohaematology cases encountered in routine clinical practice.

Methods: This study included 4 immunohaematology cases: ABO subgroup, ABO chimerism, antibody to a high-frequency antigen (HFA), and anti-CD47 interference. We designed a hybridization capture-based NGS panel targeting 39 blood group-related genes and applied it to the 4 cases.

Results: NGS analysis revealed a novel intronic variant (NM_020469.3:c.29-10T>G) in a patient with an A_el phenotype and detected a small fraction of ABO*A1.02 (approximately 3-6%) coexisting with the major genotype ABO*B.01/O.01.02 in dizygotic twins. In addition, NGS analysis found a homozygous stop-gain variant (NM_004827.3:c.376C>T, p.Gln126*; ABCG2*01N.01) in a patient with an antibody to an HFA; consequently, this patient's phenotype was predicted as Jr(a-). Lastly, blood group phenotypes predicted by NGS were concordant with those determined by serology in 2 patients treated with anti-CD47 drugs.

Conclusion: NGS-based blood group genotyping can be used for identifying ABO subgroup alleles, low levels of blood group chimerism, and antibodies to HFAs. Furthermore, it can be applied to extended blood group antigen matching for patients treated with anti-CD47 drugs.

Keywords: ABO subgroup; Anti-CD47 interference; Chimerism; Next-generation sequencing; Rare blood group.

Fig. 1

Pedigree of a family with dizygotic twins showing ABO chimerism (patients 2 and 3).

Fig. 2

Identification of a small fraction of ABO*A1.02 in dizygotic twins (patient 2, A; patient 3, B) with the major genotype ABO*B.01/O.01.02. The black arrows indicate three variants (c.261 G, c.297A, and c.467C>T) located on ABO*A1.02. The VAFs of c.297A, c.467C>T unique to ABO*A1.02 were as follows: c.297A: 5.8% for patient 2 (47 of 809 reads) and 3.8% for patient 3 (35 of 918 reads); c.467C>T: 4.8% for patient 2 (34 of 709 reads) and 3.2% for patient 3 (27 of 851 reads). The red boxes indicate that c.297A is in cis with c.261 G. *c.261 G has no genomic coordinate on hg19, as the O allele containing c.261delG is used as a reference on hg19. Therefore, the A or B allele containing c.261 G appears to have an insertion (chr9:136132908_136132909insC), whereas the O allele containing c.261delG appears to have no variant.

Fig. 3

Identification of a rare null allele (ABCG2*01N.01) in a patient with an alloantibody to an HFA. The black arrow indicates a homozygous stop-gain variant in ABCG2 exon 4 (c.376C>T, p.Gln126*).

Fig. 4

Identification of GYPA*01 by visual inspection using IGV. The red boxes indicate the reads containing all three variants on GYPA*01 (c.59C, c.71G, and c.72T in GYPA exon 2; black arrows). The number of reads containing all these variants were as follows: patient 5: 5 out of a total of 108 reads; patient 6: 1 out of a total of 97 reads.