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 5 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 6 39.8. Each sample harbored on average 43 6 9 variants, of which 10 6 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.H uman blood group antigens are of significance in transfusion medicine, because patients who have made antibodies to red blood cell antigens are at risk of being affected by hemolytic transfusion reactions after the transfusion of incompatible blood. The International Society of Blood Transfusion has defined 36 blood group systems and over 350 blood group antigens.1 Different blood group systems exhibit varying degrees of antigen polymorphism, and the clinical significance of red blood cell antibodies also varies. [2][3][4] As a minimum requirement in blood transfusion safety, all blood donors are screened for ABO and the D antigen as well as for blood group antibodies known to be clinically significant. 5The majority of antigens are missense mutations and a consequence of single nucleotide variants (SNVs); however, genetic variations, such as insertions/deletions and splice-site variants, have a qualitative and/or quantitative impact on antigen expression. Blood group systems, such as RH and MNS, exhibit an additional layer of genetic variation. These arise because each system comprises homologous genes in which gene crossover or g...

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.

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 (DW) 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 (DW)+ and Rh32–. CONCLUSION A novel RHD*D‐CE(5‐6)‐D hybrid allele encodes a partial RhD epitope and carries the LFA RH23 (DW). 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 (DW)+ cells to RH23 (DW)– recipients also pose an alloimmunization risk.

The noninvasive prenatal determination of fetal RhD blood group can be achieved by analyzing cell-free fetal DNA in maternal plasma. An RhD-positive fetus can be identified by detecting the presence of RHD gene sequences in the plasma of an RhD-negative woman who does not possess the gene. 1Many research groups have demonstrated the high diagnostic accuracy of this approach, and the test has now been available as a clinical service in many laboratories in Europe and the USA.In addition to the complete deletion of the RHD gene, a number of RHD gene variants that consist of single-nucleotide mutations have been reported to be associated with RhDnegative or the DEL phenotype, that is, a very weak expression of the D antigen. Pregnant women who harbor RHD variants, such as ones with the RHD(IVS3+1G>A) mutation, are capable of producing anti-D alloantibodies and thus are at risk for hemolytic disease of the fetus and newborn (HDFN).2,3 Thus far, noninvasive prenatal RHD genotyping for women carrying such intact but dysfunctional RHD variants has not been reported. The challenge is due to the high background of maternal DNA in maternal plasma and fetal DNA levels amount to only 10% of the total plasma DNA molecules. 4 For pregnant women with a complete RHD gene deletion, the fetal RHD DNA detection is free from maternal background interference because the whole gene is absent in the maternal genome. On the other hand, for maternal plasma involving maternal RHD variants, one has to discriminate the fetal-allele sequences among the high background of maternal-allele sequences that differ from each other by a single nucleotide only. Digital polymerase chain reaction (PCR) has been shown to be a sensitive and specific tool for detecting single nucleotide variations between the mother and the fetus in maternal plasma. 5 In this study, we investigated the feasibility of using digital PCR to determine the fetal inheritance of RHD allele noninvasively using maternal plasma. As proof-of-principle, we investigated two alloimmunized pregnant women carrying the RHD(IVS3+1G>A) mutation. 3This investigation was triggered after two separate obstetric cases that were both Caucasian, phenotyped as RhD-negative by hospital serology, and were sensitized to the D antigen. Maternal plasma samples were referred to the blood service for noninvasive prenatal RHD genotyping. However, the fetal RHD typing by real-time PCR was unsuccessful due to the presence of maternal RHD allele sequences in maternal plasma. The RHD dosage analysis of maternal genomic DNA by PCR 6 showed that the two women were hemizygous for RHD, and they carried the DEL associated RHD*IVS3+1G>A allele as detected using a commercial single nucleotide polymorphism (SNP) based blood group genotyping platform (BLOODchip micorarray, Progenika Biopharma S.A.). The two women were confirmed serologically to express the DEL

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