Blood Group Genomics
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BLOOD GROUP GENOMICS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of 36 blood group alleles among 396 Thai samples reveals region-specific variants Philaiphon Jongruamklang,1 Christoph Gassner,2 Stefan Meyer,2 Aksarakorn Kummasook,3 Marion Darlison,1 Chayanun Boonlum,4 Surin Chanta,5 Beat M. Frey,2 Martin L. Olsson,1,6* and Jill R. Storry 1,6* lood group antigen polymorphism shows great BACKGROUND: Blood group phenotype variation has variation in different world populations. The been attributed to potential resistance to pathogen reason for this is not completely understood; invasion. Variation was mapped in blood donors from however, it has been attributed to both Lampang (northern region) and Saraburi (central region), B Thailand, where malaria is endemic. The previously unknown blood group allele profiles were characterized ABBREVIATIONS: MALDI-TOF MS 5 matrix-assisted laser and the data were correlated with phenotypes. The high desorption/ionization time-of-flight mass spectrometry; PCR- incidence of the Vel-negative phenotype previously ASP 5 polymerase chain reaction with allele-specific reported in Thais was investigated. primers; SNP(s) 5 single nucleotide polymorphism(s). STUDY DESIGN AND METHODS: DNA from 396 From 1Hematology and Transfusion Medicine, Department of blood donors was analyzed by matrix-assisted laser Laboratory Medicine, Lund University, Lund, Sweden; desorption/ionization–time-of-flight mass spectrometry. 2Molecular Diagnostics & Research (MOC), Blood Transfusion Outliers were investigated by serology and DNA Service Zurich,€ Zurich-Schlieren,€ Switzerland; 3Department of sequencing. Allele discrimination assays for SMIM1 Medical Technology, School of Allied Health Sciences, rs1175550A/G and ACKR1 rs118062001C/T were University of Phayao, Phayao, Thailand; 4Transfusion Medicine, performed and correlated with antigen expression. Department of Medical Technology and Clinical Laboratory, RESULTS: All samples were phenotyped for Rh, MNS, Saraburi Hospital, Saraburi, Thailand; 5Transfusion Medicine, and K. Genotyping/phenotyping for RhD, K, and S/s Department of Medical Technology and Clinical Laboratory, showed 100% concordance. Investigation of three RHCE Lampang Hospital, Lampang, Thailand; and 6Clinical outliers revealed an e-variant antigen encoded by Immunology and Transfusion Medicine, Laboratory Medicine, RHCE*02.22. Screening for rs147357308 (RHCE Office of Medical Services, Lund, Sweden. c.667T) revealed a frequency of 3.3%. MN typing Address reprint requests to: Jill R. Storry, PhD, Clinical discrepancies in 41 samples revealed glycophorin Immunology and Transfusion Medicine, Akutgatan 8, SE-22185 variants, of which 40 of 41 were due to Mia. Nine samples Lund, Sweden; e-mail: [email protected]. (2.3%) were heterozygous for FY*01W.01 (c.265C > T), This work was supported by governmental grants for clini- and six samples (1.5%) were heterozygous for cal research to university healthcare in Lund, Sweden (ALF- JK*02N.01. All samples were wildtype SMIM1 SKANE-446521 to MLO); the Knut and Alice Wallenberg homozygotes with 97% homozygosity for rs1175550A. Foundation (2014.0312 to MLO), the Swedish Research Council CONCLUSIONS: Matrix-assisted laser desorption/ (2014-71X-14251 to MLO) and the Crafoord Foundation ionization–time-of-flight mass spectrometry is an efficient (20151060 to JRS). PJ is supported by an education grant from method for rapid routine genotyping and investigation of the University of Phayao, Thailand. outliers identified novel variation among our samples. *Both these authors contributed equally. The expected high prevalence of the Mi(a1) phenotype Received for publication December 27, 2017; revision was observed from both regions. Of potential clinical received February 24, 2018; and accepted February 26, 2018. relevance in a region where transfusion-dependent doi:10.1111/trf.14624 thalassemia is common, we identified two RHCE*02 VC 2018 AABB alleles known to encode an e-variant antigen. TRANSFUSION 2018;58;1752–1762 1752 TRANSFUSION Volume 58, July 2018 THAI BLOOD GROUPS BY MASS SPECTROMETRY migration and potential resistance to different pathogens. genotyping platform since 201314 and has been proven to Many blood group antigens have been shown to be spe- be a reliable and accurate predictor of blood group phe- cific receptors for various pathogens.1,2 Specifically, notype, although mainly in white populations in Europe. malaria has been described as one of the major driving We chose the MALDI-TOF MS platform to character- forces of human erythroid variation.3,4 Impairments to ize the blood group allele profile of 396 Thai blood donors structural proteins such as SLC4A1 (band 3; Southeast by, and correlated the results with RhD, RhCE, MNS, K/k Asian ovalocytosis), and glycophorin C (elliptocytosis in phenotype, as well as resolved apparent discrepancies. We the Gerbich and Yus phenotypes), globin gene defects targeted known and potential malaria receptors, for exam- (thalassemias, sickle cell disease), are all attributed to ple, ACKR1, MNS, Vel, and CD147, and looked for further attempts to evade or minimize infection by Plasmodium polymorphism among our cohort. spp., as is the absence of ACKR1 in the Fy(a–b–) pheno- type and the generation of hybrid proteins in the MNS MATERIALS AND METHODS system.3,4 Malaria is endemic across the equatorial belt and encompasses a large swath of Southeast Asia. In Thai- Samples land, malaria is due primarily to infection by Plasmodium Acid-citrate-dextrose–anticoagulated blood samples were falciparum (44%) and Plasmodium vivax (47%), although obtained following informed consent and approval from other Plasmodium spp. are accountable for the remaining 398 unrelated blood donors at two blood banks in Thai- 9%, including Plasmodium knowlesi, recently described as land: 199 samples were collected at Lampang Hospital 5 a human pathogen. P.falciparum is known to use a num- located in the north (of which two samples hemolyzed ber of different red blood cell (RBC) ligands for attach- during transit; valid 197), and 199 samples were collected ment and invasion, whereas P. vivax uses ACKR1 at Saraburi Hospital located in central Thailand. Aliquots 6,7 exclusively. For these reasons, we wanted to map varia- of all samples were frozen (1) in a RNA-stabilizing reagent tion in blood group distribution in two groups of blood (TRIzol LS, ThermoFisher Scientific) for subsequent RNA donors from different regions of Thailand where malaria analysis; (2) as buffy coats for downstream genomic analy- is endemic, one northern (Lampang) and one central sis; (c) RBCs in glycerol (1:1; S.A.L.F. S.p.A. Laboratorio (Saraburi). Farmacologico); and (4) plasma. Phenotypic studies of Thai blood donors have been reported previously. As early as 1967, Chandanayingyong DNA preparation and colleagues8 studied the blood groups of 456 blood Genomic DNA was extracted from 396 whole blood sam- donors and were among the first to describe the high ples using a silica-membrane–based nucleic acid purifica- prevalence of the Mi(a1) phenotype. Intriguingly, they tion system (QIAsymphony DSP DNA Mini Kit, Qiagen, observed an unexpectedly high prevalence of the Vel- Inc.) on a modular instrument (QIAsymphony SP, Qiagen, negative phenotype. A retrospective review of the sero- Inc.), as recommended by the manufacturer. Quantity and logic blood grouping records of more than a million blood quality of DNA samples were measured by spectropho- donations from the Thai National Blood Center was reported in 2002 by Fongsarun and colleagues.9 From tometry (Nanodrop Technologies, Inc.). their results, the allele frequencies were calculated for the common blood group antigens. Again, a high prevalence MALDI-TOF MS of Mia was observed (9.1%) but the results were otherwise Genomic DNA was analyzed by a MALDI-TOF MS plat- unremarkable. The Mi(a1) phenotype has been studied form (MassARRAY, Agena Bioscience GmbH) that targets more closely both by serologic and molecular techniques. 36 blood group–related single nucleotide polymorphisms Kaset and colleagues10 used the molecular screening tech- (SNPs) in 15 blood group systems, as described 14,15 nique first described by Palacajornsuk and coworkers.11 to previously. characterize 107 Mi(a1) blood donor samples and identi- Briefly, the platform includes two in-house multiplex fied five different MNS hybrid genes among these donors. reactions, allowing the parallel genotyping of 23 and 13 Blood group genotyping as a method for predicting SNPs, respectively, in two single tubes per DNA sample. the blood group phenotype is now a well-accepted prac- Test reagents were purchased as a kit (Complete iPLEX Pro tice, especially in larger blood centers where the aim is to Genotyping Reagent Set, Agena Bioscience GmbH). Multi- increase the number of well-characterized blood units plex reaction primers were purchased from Metabion as and enable better inventory matching and management described previously.14 TheSNPstargetedbytheplatform for patients with antibodies and patients at risk of are specific for the discrimination of (1) the wildtype versus alloimmunization.12,13 c.261delG variation of ABO; (2) the erythrocyte antigens Matrix-assisted laser desorption/ionization time-of- RhC/c, RhCw,RhE/e,K/k,Kpa/b,Jsa/b,Jka/b,Fya/b,M/N,S/s, a/b flight mass spectrometry (MALDI-TOF MS) has been and Do ; (3) the null phenotypes K0 (KEL*02N.01, developed and used as a high-throughput blood group KEL*02N.06), Jk(a–b–) (JK*02N.01, JK*02N.02,