Molecular typing for the Indian blood group associated 252G>C single nucleotide polymorphism in a selected cohort of Australian blood donors |
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Authors: | Genghis H. Lopez Rhiannon S. McBean Brett Wilson Darryl L. Irwin Yew-Wah Liew Catherine A. Hyland Robert L. Flower |
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Affiliation: | 1.Research and Development Division, Australian Red Cross Blood Service, Queensland, Australia;2.Red Cell Reference Laboratory, Australian Red Cross Blood Service, Queensland, Australia;3.Sequenom Inc. Asia Pacific, Herston, Queensland, Australia |
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Abstract: | BackgroundThe Indian blood group antigens, Ina and Inb, are clinically significant in transfusion medicine. However, antisera to type these antigens are difficult to obtain. The Inb antigen is a high frequency antigen present in all populations, while the frequency of the antithetical Ina ranges from 0.1% in Caucasians up to 11% in Middle Eastern groups. This antigen polymorphism is encoded by the single nucleotide polymorphism (SNP) 252G>C in CD44. The aim of this study was to establish and compare two genotyping methods to measure the frequency of the IN*A and IN*B alleles in a blood donor cohort.Materials and methodsDonor blood samples (n=151) were genotyped by a novel real-time polymerase chain reaction (PCR) high-resolution meltcurve (HRM) analysis and a custom matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) assay. Samples with the rare IN*A allele were further investigated by nucleotide sequencing, red cell agglutination, and flow cytometry techniques.ResultsIn this study group, 149 IN*B homozygous and 2 IN*A/B heterozygous samples were detected with 100% concordance between HRM and MALDI-TOF MS methods. For PCR HRM, amplicon melting alone did not differentiate IN*A and IN*B alleles (class 3 SNP), however, the introduction of an unlabelled probe (UP) increased the resolution of the assay. Sequencing confirmed that the two non-homozygous samples were IN*A/B heterozygous and phenotyping by red cell agglutination, and flow cytometry confirmed both Ina and Inb antigens were present as predicted.DiscussionGenotyping permits conservation of rare antisera to predict blood group antigen phenotype. In PCR UP-HRM the IN*A and IN*B alleles were discriminated on the basis of their melting properties. The Ina frequency in this selected donor population was 1.3%. Application of genotyping methods such as these assists in identifying donors with rare blood group phenotypes of potential clinical significance. |
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Keywords: | Indian blood group genotyping high-resolution melting analysis MALDI-TOF MS |
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