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 Immunohematology reference laboratories provide red blood cell (RBC), platelet (PLT), and neutrophil typing to resolve complex cases, using serology and commercial DNA tests that define clinically important antigens. Broad‐range exome sequencing panels that include blood group targets provide accurate blood group antigen predictions beyond those defined by serology and commercial typing systems and identify rare and novel variants. The aim of this study was to design and assess a panel for targeted exome sequencing of RBC, PLT, and neutrophil antigen–associated genes to provide a comprehensive profile in a single test, excluding unrelated gene targets. Study Design and Methods An overlapping probe panel was designed for the coding regions of 64 genes and loci involved in gene expression. Sequencing was performed on 34 RBC and 17 PLT/neutrophil reference samples. Variant call outputs were analyzed using software to predict star allele diplotypes. Results were compared with serology and previous sequence genotyping data. Results Average coverage exceeded 250×, with more than 94% of targets at Q30 quality or greater. Increased coverage revealed a variant in the Scianna system that was previously undetected. The software correctly predicted allele diplotypes for 99.5% of RBC blood groups tested and 100% of PLT and HNA antigens excepting HNA‐2. Optimal throughput was 12 to 14 samples per run. Conclusion This single‐test system demonstrates high coverage and quality, allowing for the detection of previously overlooked variants and increased sample throughput. This system has the potential to integrate genomic testing across laboratories within hematologic reference settings.
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.
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