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Background: We report an obstetric case involving an RhD-positive woman who had developed a red blood cell (RBC) antibody that was not detected until after delivery of a newborn, who presented with a positive direct antiglobulin test result. Immunohematology studies suggested that the maternal antibody was directed against a low-prevalence antigen on the paternal and newborn RBCs., Results: Comprehensive blood group profiling by targeted exome sequencing revealed a novel nonsynonymous single nucleotide variant (SNV) RHCE c.486C>G (GenBank MZ326705) on the RHCE*Ce allele, for both the father and newborn. A subsequent genomic-based study to profile blood groups in an Indigenous Australian population revealed the same SNV in 2 of 247 individuals. Serology testing showed that the maternal antibody reacted specifically with RBCs from these two individuals., Discussion: The maternal antibody was directed against a novel antigen in the Rh blood group system arising from an RHCE c.486C>G variant on the RHCE*Ce allele linked to RHD*01. The variant predicts a p.Asn162Lys change on the RhCE protein and has been registered as the 56th antigen in the Rh system, ISBT RH 004063., Conclusion: This antibody was of clinical significance, resulting in a mild to moderate hemolytic disease of the fetus and newborn (HDFN). In the past, the cause of such HDFN cases may have remained unresolved. Genomic sequencing combined with population studies now assists in resolving such cases. Further population studies have potential to inform the need to design population-specific red cell antibody typing panels for antibody screening in the Australian population., (© 2022 AABB.)

Background: Low-prevalence antigen s D (MNS23) is encoded by GYPB c.173C > G. Hemolytic disease of the fetus and newborn (HDFN) due to anti-s D is rare. A mother delivered a newborn whose red blood cells (RBCs) were DAT-positive and was later diagnosed with HDFN. Serum from the mother was incompatible with the father's RBCs and was used to screen 184 Thai blood donors. This study aimed to investigate the cause of HDFN in a Thai family and determine the prevalence of s D in Thai blood donors., Materials and Methods: Three family members and four blood donors were investigated in the study. Massively Parallel Sequencing (MPS) was used for genotyping. Standard hemagglutination techniques were used in titration studies, phenotyping, and enzyme/chemical studies. Anti-s, anti-Mi a , anti-JENU, and anti-s D reagents were used in serological investigations., Results: The mother was GYP*Mur/Mur. The father and the four donors were GYPB*s/s D predicting S - s + s D +. The baby was GYP*Mur/sD and his RBCs were Mi a +, s +  w with anti-s (P3BER) and JENU+ w . RBCs from two GYPB*s D -positive blood donors reacted with anti-s D (Dreyer). Proteolytic enzyme α-chymotrypsin-treated s D + cells did not react with anti-s D (Wat) produced by the GP.Mur/Mur mother but reacted with the original anti-s D (Dreyer)., Discussion: This is the first report of HDFN due to anti-s D in the Asian population. The genotype frequency for GYPB*s D in a selected Thai blood donor population is 2.2% (4/184). Anti-s D should be considered in mothers with Southeast Asian or East Asian background when antibody identification is unresolved in pregnancies affected by HDFN., (© 2022 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Background: MNS hybrid GP(B-A-B) glycophorins are more commonly found in Southeast Asians and alloantibodies to antigens they carry are clinically significant. Detection of hybrid glycophorins by serologic techniques is limited due to lack of commercial reagents. In this study, a genotyping method for GP(B-A-B) hybrid glycophorins based on high-resolution melting (HRM) analysis was applied for genotyping analysis in the Chinese Southern Han population., Study Design and Methods: DNA samples from 3104 Chinese Southern Han blood donors were collected. GYP(B-A-B) genotypes were analyzed by HRM assay. Parts of samples (n = 106) were also tested by multiplex ligation-dependent probe amplification (MLPA) assay. Direct sequencing was conducted in samples with variant melting curve profiles., Results: A total of five GYP(B-A-B) genotypes (201/3104, 6.5%) were identified, which were GYP*Mur heterozygote (n = 194), GYP*Mur homozygote (n = 3), GYP*Bun heterozygote (n = 2), GYP*HF heterozygote (n = 1), and a novel GYP(B-A-B) hybrid allele (n = 1). Genotyping results for GYP*Mur and wild-type GYPB samples obtained by HRM were consistent with MLPA, while GYP*Bun and GYP*HF heterozygote identified by HRM could only be identified to have one copy of 5' inactive splice site of GYPB Pseudoexon 3 by MLPA. In addition, 10 single-nucleotide polymorphisms (SNPs) including four known and six novel SNPs were identified in 31 samples. One sample was identified carrying both GYP*Mur and GYP*Sch alleles., Conclusion: The HRM assay could distinguish the GYP(B-A-B) hybrid alleles successfully. Polymorphisms identified within the GYPB gene should be taken into consideration when developing GYP(B-A-B) genotyping kits for the Chinese population., (© 2018 AABB.)

Background: The RhD blood group antigen is extremely polymorphic and the DEL phenotype represents one such class of polymorphisms. The DEL phenotype prevalent in East Asian populations arises from a synonymous substitution defined as RHD*1227A. However, initially, based on genomic and cDNA studies, the genetic basis for a DEL phenotype in Taiwan was attributed to a deletion of RHD Exon 9 that was never verified at the genomic level by any other independent group. Here we investigate the genetic basis for a Caucasian donor with a DEL partial D phenotype and compare the genomic findings to those initial molecular studies., Study Design and Methods: The 3'-region of the RHD gene was amplified by long-range polymerase chain reaction (PCR) for massively parallel sequencing. Primers were designed to encompass a deletion, flanking Exon 9, by standard PCR for Sanger sequencing. Targeted sequencing of exons and flanking introns was also performed., Results: Genomic DNA exhibited a 1012-bp deletion spanning from Intron 8, across Exon 9 into Intron 9. The deletion breakpoints occurred between two 25-bp repeat motifs flanking Exon 9 such that one repeat sequence remained., Conclusion: Deletion mutations bordered by repeat sequences are a hallmark of slipped-strand mispairing (SSM) event. We propose this genetic mechanism generated the germline deletion in the Caucasian donor. Extensive studies show that the RHD*1227A is the most prevalent DEL allele in East Asian populations and may have confounded the initial molecular studies. Review of the literature revealed that the SSM model explains some of the extreme polymorphisms observed in the clinically significant RhD blood group antigen., (© 2017 AABB.)

Background: We previously demonstrated that targeted exome sequencing accurately defined blood group genotypes for reference panel samples characterized by serology and single-nucleotide polymorphism (SNP) genotyping. Here we investigate the application of this approach to resolve problematic serology and SNP-typing cases., Study Design and Methods: The TruSight One sequencing panel and MiSeq platform was used for sequencing. CLC Genomics Workbench software was used for data analysis of the blood group genes implicated in the serology and SNP-typing problem. Sequence variants were compared to public databases listing blood group alleles. The effect of predicted amino acid changes on protein function for novel alleles was assessed using SIFT and PolyPhen-2., Results: Among 29 unresolved samples, sequencing defined SNPs in blood group genes consistent with serologic observation: 22 samples exhibited SNPs associated with varied but known blood group alleles and one sample exhibited a chimeric RH genotype. Three samples showed novel variants in the CROM, LAN, and RH systems, respectively, predicting respective amino acid changes with possible deleterious impact. Two samples harbored rare variants in the RH and FY systems, respectively, not previously associated with a blood group allele or phenotype. A final sample comprised a rare variant within the KLF1 transcription factor gene that may modulate DNA-binding activity., Conclusion: Targeted exome sequencing resolved complex serology problems and defined both novel blood group alleles (CD55:c.203G>A, ABCB6:c.1118_1124delCGGATCG, ABCB6:c.1656-1G>A, and RHD:c.452G>A) and rare variants on blood group alleles associated with altered phenotypes. This study illustrates the utility of exome sequencing, in conjunction with serology, as an alternative approach to resolve complex cases., (© 2017 AABB.)

Background: Individuals with the partial D phenotype when exposed to D+ red blood cells (RBCs) carrying the epitopes they lack may develop anti-D specific for the missing epitopes. DNB is the most common partial D in Caucasians and the clinical significance for anti-D in these individuals is unknown., Study Design and Methods: This article describes the serologic genotyping results and clinical manifestations in two group D+ babies of a mother presenting as group O, D+ with alloanti-D., Results: The mother was hemizygous for RHD*DNB gene and sequencing confirmed a single-nucleotide change at c.1063G>A. One baby (group A, D+) displayed bilirubinemia at birth with a normal hemoglobin level. Anti-A and anti-D were eluted from the RBCs. For the next ongoing pregnancy, the anti-D titer increased from 32 to 256. On delivery the baby typed group O and anti-D was eluted from the RBCs. This baby at birth exhibited anemia, reticulocytosis, and hyperbilirubinemia requiring intensive phototherapy treatment from Day 0 to Day 9 after birth and was discharged on Day 13. Intravenous immunoglobulin was also administered. Both babies were heterozygous for RHD and RHD*DNB., Conclusion: The anti-D produced by this woman with partial D DNB resulted in a case of hemolytic disease of the fetus and newborn (HDFN) requiring intensive treatment in the perinatal period. Anti-D formed by women with the partial D DNB phenotype has the potential to cause HDFN where the fetus is D+. Women carrying RHD*DNB should be offered appropriate prophylactic anti-D and be transfused with D- RBCs if not already alloimmunized., (© 2017 AABB.)

Background: Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS., Study Design and Methods: Donor samples (n = 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems., Results: The average sequencing depth per target region was 66.2 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement., Conclusion: The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting., (© 2017 AABB.)

Background: Blood donors whose red blood cells (RBCs) exhibit a partial RhD phenotype, lacking some D epitopes, present as D+ in routine screening. Such phenotypes can exhibit low-frequency antigens (LFAs) of clinical significance. The aim of this study was to describe the serologic and genetic profile for a blood donor with an apparent D+ phenotype carrying a variant RHD gene where D Exons 5 and 6 are replaced by RHCE Exon (5-6)., Study Design and Methods: Anti-D monoclonal antibodies were used to characterize the presentation of RhD epitopes on the RBCs. RHD exon scanning and DNA sequencing of short- and long-range polymerase chain reaction amplicons were used to determine the RHD structure and sequence. Extended phenotyping for LFAs RH23 (D(W) ) and Rh32 was performed., Results: The donor serology profile was consistent with partial RhD epitope presentation. The donor was hemizygous for an RHD variant allele described as RHD*D-CE(5-6)-D hybrid. The RHCE gene insert is at least 3.868 kb with 5' and 3' breakpoints between IVS4 + 132-c.667 and IVS6 + 1960-IVS6 + 2099, respectively. The sequence for this hybrid was assigned GenBank Accession Number KT099190.2. The RBCs were RH23 (D(W) )+ and Rh32-., Conclusion: A novel RHD*D-CE(5-6)-D hybrid allele encodes a partial RhD epitope and carries the LFA RH23 (D(W) ). This and the epitope profile resemble the partial DVa phenotype. Given that RBCs from this individual lack some RhD epitopes, there is an alloimmunization risk if the donor is exposed to D+ RBCs. Conversely, transfusions of RH23 (D(W) )+ cells to RH23 (D(W) )- recipients also pose an alloimmunization risk., (© 2016 AABB.)

Background: The introduction of molecular methods into routine blood typing is prompting the identification of new blood group alleles. Discrepancies between the results of genotyping and serology or chance events uncovered during genotyping prompted additional investigations, which revealed six new RHCE variant alleles., Study Design and Methods: Samples from eight blood donors, two patients (one prenatal), and a patient's relative, all of diverse racial origin, were analyzed by standard serology methods, targeted genotyping arrays, DNA sequencing, and allele-specific polymerase chain reaction., Results: Six new RHCE alleles were identified, namely, RHCE*cE84A, RHCE*ce202G, RHCE*ce307T, RHCE*Ce377G, RHCE*ce697G,712G,733G,744C, and RHCE*Ce733G., Conclusion: While implementation of new assays in commercial genotyping platforms to detect the polymorphisms reported here may not be justified given their apparent rarity, software interpretative algorithms may benefit from the identification of new alleles for a more accurate determination of genotypes and prediction of phenotypes., (© 2015 AABB.)