IMMUNOHEMATOLOGY Targeted exome sequencing defines novel and rare variants in complex blood group serology cases for a red blood cell reference laboratory setting Elizna M. Schoeman ,1 Eileen V. Roulis ,1 Yew-Wah Liew,2 Jacqueline R. Martin,2 Tanya Powley,2 Brett Wilson,2 Glenda M. Millard ,1 Eunike C. McGowan ,1 Genghis H. Lopez ,1 Helen O’Brien ,1 Jennifer A. Condon,3 Robert L. Flower ,1 and Catherine A. Hyland 1 he key role of a modern red blood cell (RBC) ref- BACKGROUND: We previously demonstrated that erence laboratory in transfusion medicine is to targeted exome sequencing accurately defined blood employ serology to solve complex problems. group genotypes for reference panel samples Often, the lack of appropriate RBCs, sera, or characterized by serology and single-nucleotide T polymorphism (SNP) genotyping. Here we investigate reagents makes confident resolution of a complex case the application of this approach to resolve problematic difficult. Resources can be costly and scarce, if available at serology and SNP-typing cases. all, and often require collaboration and generosity of col- 1 STUDY DESIGN AND METHODS: The TruSight One leagues from other international laboratories. In addition, sequencing panel and MiSeq platform was used for reference laboratories commonly employ single- sequencing. CLC Genomics Workbench software was nucleotide polymorphism (SNP) typing microarrays for used for data analysis of the blood group genes RBC genotyping investigations; however, these arrays are implicated in the serology and SNP-typing problem. not comprehensive in their coverage of blood groups or 2-5 Sequence variants were compared to public databases variants. When the combination of traditional serologic listing blood group alleles. The effect of predicted amino methods and typing by SNP microarray fails to resolve, acid changes on protein function for novel alleles was assessed using SIFT and PolyPhen-2. ABBREVIATIONS: ExAC 5 Exome Aggregation Consortium; RESULTS: Among 29 unresolved samples, sequencing MPS 5 massively parallel sequencing; RCRL 5 Red Cell defined SNPs in blood group genes consistent with Reference Laboratory; SNP(s) 5 single-nucleotide serologic observation: 22 samples exhibited SNPs polymorphism(s); SNV(s) 5 single-nucleotide variant(s); associated with varied but known blood group alleles and TSO 5 TruSight One. one sample exhibited a chimeric RH genotype. Three From 1Clinical Services and Research; and the 2Red Cell samples showed novel variants in the CROM, LAN, and Reference Laboratory, Australian Red Cross Blood Service, RH systems, respectively, predicting respective amino Kelvin Grove, Queensland, Australia; and the 3Red Cell acid changes with possible deleterious impact. Two Reference Laboratory, Australian Red Cross Blood Service, samples harbored rare variants in the RH and FY Melbourne, Victoria, Australia. systems, respectively, not previously associated with a Address reprint requests to: Catherine A. Hyland, PhD, blood group allele or phenotype. A final sample MSc, Clinical Services and Research, Australian Red Cross comprised a rare variant within the KLF1 transcription Blood Service, 44 Musk Avenue, Kelvin Grove, Queensland factor gene that may modulate DNA-binding activity. 4059, Australia; e-mail: [email protected]. CONCLUSION: Targeted exome sequencing resolved The Australian government funds the Australian Red Cross complex serology problems and defined both novel blood Blood Service for the provision of blood, blood products, and group alleles (CD55:c.203G>A, ABCB6:c.1118_1124del services to the Australian community. CGGATCG, ABCB6:c.1656-1G>A, and RHD:c.452G>A) Received for publication February 21, 2017; revision and rare variants on blood group alleles associated with al received August 21, 2017; and accepted September 5, 2017. tered phenotypes. This study illustrates the utility of exome doi:10.1111/trf.14393 sequencing, in conjunction with serology, as an alternative VC 2017 AABB approach to resolve complex cases. TRANSFUSION 2018;58;284–293 284 TRANSFUSION Volume 58, February 2018 EXOME SEQUENCING FOR COMPLEX BLOOD GROUPS further avenues of investigation are required. For some elution techniques were used to confirm presence of the reference laboratories the test algorithm at this stage will antigen. Cell separation of a dual population (Sample 7) include Sanger sequencing of targeted genes. was performed by agglutinating the D1 cells with anti-D As an alternative approach, massively parallel and separating from the unagglutinated cells using a 20% sequencing (MPS—also known as next-generation albumin gradient. sequencing) technologies are becoming more widely used Antibody identification was performed using stan- in blood banking and hematologic investigation, due to dard serologic techniques. Routinely, a commercial 11-cell the capability of identifying all variants in target blood panel (Seqirus) and autologous cells are tested by three group genes. A recent proof-of-principle study demon- tube methods: saline room temperature (248C), polyethyl- strated the capability of using whole genome sequencing ene glycol indirect antiglobulin test (IAT), and papain IAT. data for comprehensive antigen prediction of RBCs and Results from this routine testing directed further serologic platelets.6 A targeted exome sequencing approach has investigation with various enzymes or chemical modifica- been recognized for its utility in blood group genotyping tion of cells, rare phenotype cells, cord cells, antibody and has been predicted to facilitate investigation of novel adsorptions-elutions, and peptide inhibitions. blood group alleles.7,8 While studies have shown proof of principle for blood group genotyping by MPS, no studies SNP genotyping in the reference laboratory have investigated to date its clinical application to resolve Genomic DNA was extracted from fresh EDTA whole complex cases in a reference laboratory setting.6,8-12 blood samples using the DNA blood mini kit (QIAamp Previously, we validated the use of a commercially DSP, QIAGEN) for use with a robotic workstation for auto- available targeted exome sequencing panel for investiga- mated purification of DNA, RNA, or proteins (QIACube, tion of genetic variation within 28 protein-based blood QIAGEN) or manually as per the manufacturer’s instruc- group systems on a collection of well-characterized refer- tions if required. The DNA was tested using a high- ence panel samples.13 Our analyses confirmed the pheno- throughput molecular assay (BioArray BeadChip technol- types obtained using SNP microarray and serologic ogy, Immucor) for the simultaneous detection of a wide methods, as well as identifying a number of variants con- range of SNPs affecting RBC antigen expression, using a sidered “noninformative”—that is, they may be common multiplexed molecular assay (either Immucor PreciseType population variants or variants that have not previously HEA molecular BeadChip test, herein referred to as “HEA” been associated with established blood group alleles or or the RHD Molecular BeadChip test). phenotypes. The HEA test predicts antigen phenotype for RH (C, Here we investigate the potential application for c, E, e, V, VS), KEL (K, k, Kpa,Kpb,Jsa,Jsb), FY (Fya,Fyb, blood group genotyping using targeted exome sequencing GATA, Fyx), JK (Jka,Jkb), MNS (M, N, S, s, Uvar, Uneg), LU to resolve complex cases, where sequencing was (Lua,Lub), DO (Doa,Dob,Hy,Joa), LW (LWa,LWb), DI (Dia, b a b 14 requested by the Australian Red Cross Blood Service Red Di ), CO (Co ,Co ), and SC (Sc1, Sc2). The RHD test uti- Cell Reference Laboratory (RCRL). These requests were lizes 35 genetic markers associated with D phenotypes to 15 made either where serology typing was discordant to SNP make RHD variant calls. genotyping or where confirmation was needed to support antigen typing. MPS library preparation and sequencing We prepared DNA libraries for sequencing using a MATERIALS AND METHODS sequencing panel (TruSight One [TSO], Illumina) as per manufacturer’s instructions. The TSO panel amplifies RCRL serology investigations exons and untranslated regions for 4813 clinically signifi- Samples referred to investigate the D status were first cant genes, including the 41 genes related to 33 blood tested with two anti-D antisera: one, Epiclone-2 anti-D group systems and genes for two transcription factors, (IgM) (Seqirus) that does not detect most partial D and KLF1 and GATA1 (Table S1, available as supporting infor- weak D types, and another, anti-D (monoclonal blend) mation in the online version of this paper). The genes for Gamma-clone (Immucor) that does detect most partial D three (JMH, VEL, and XG) of the 36 blood group systems and weak D types. Samples were further tested with were excluded by the manufacturer of the kit. MPS was ALBAclone advanced partial Rh(D) typing kit (Alba Biosci- performed on the MiSeq sequencing platform (Illumina) ence) as per the manufacturer’s instruction. to generate paired-end 150-bp reads as described 13 Phenotyping was performed, where possible, with previously. commercially available antisera. When commercial anti- sera were not available, in-house antisera was used with Data analysis the optimum technique for that particular antiserum. FASTQ files were generated by the MiSeq system on- Where weak antigen expression was suspected, adsorption- instrument software and exported to computer software Volume 58, February 2018 TRANSFUSION 285 SCHOEMAN ET AL. (CLC Genomics Workbench software, Version 8.5, QIA- GEN) for secondary