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Genotyping for Glycophorin GYP(B-A-B) Hybrid Genes Using a Single Nucleotide Polymorphism-Based Algorithm by Matrix-Assisted Laser Desorption/Ionisation, Time-of-Flight Mass Spectrometry

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Abstract

The genetic basis for five GP(B-A-B) MNS system hybrid glycophorin blood group antigens results from rearrangement between the homologous GYPA and GYPB genes. Each hybrid glycophorin displays a characteristic profile of antigens. Currently, no commercial serological reagents are currently available to serologically type for these antigens. The aim of this study was to develop a single nucleotide polymorphism (SNP) mapping genotyping technique to allow characterisation of various GYP(B-A-B) hybrid alleles. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) assays were designed to genotype five GYP(B-A-B) hybrid alleles. Eight nucleotide positions were targeted and incorporated into the SNP mapping protocol. The allelic frequencies were calculated using peak areas. Sanger sequencing was performed to resolve a GYP*Hop 3′ breakpoint. Observed allelic peak area ratios either coincided with the expected ratio or were skewed (above or below) from the expected ratio with switching occurring at and after the expected break point to generate characteristic mass spectral plots for each hybrid. Sequencing showed that the GYP*Hop crossover in the intron 3 region, for this example, was identical to that for GYP*Bun reference sequence. An analytical algorithm using MALDI-TOF MS genotyping platform defined GYPA inserts for five GYP(B-A-B) hybrids. The SNP mapping technique described here demonstrates proof of concept that this technology is viable for genotyping hybrid glycophorins, GYP(A-B-A), GYP(A-B) and GYP(B-A), and addresses the gap in current typing technologies.

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Acknowledgments

Australian governments fund the Australian Red Cross Blood Service for the provision of blood, blood products and services to the Australian community. We would like to thank Dr Elizna Schoeman for help us with the Sanger Sequencing chromatogram figure. We are grateful to Dr Makoto Uchikawa of the Japanese Red Cross, Tokyo, Japan for providing us DNA samples for GYP*HF. The work was supported by Science and Technology Project of Guangzhou City (No.201509010009).

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    Authors and Affiliations

    1. Clinical Services and Research, Australian Red Cross Blood Service, 44 Musk Avenue, Kelvin Grove, QLD, 4059, Australia

      Ling Wei, Genghis H. Lopez, Catherine A. Hyland & Robert L. Flower

    2. Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, People’s Republic of China

      Ling Wei, Yanli Ji & Guangping Luo

    3. Red Cell Reference Laboratory, Australian Red Cross Blood Service, 100-154 Batman St., West Melbourne, VIC, 3003, Australia

      Jennifer A. Condon

    4. Agena Bioscience, Herston, QLD, 4006, Australia

      Darryl L. Irwin

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    1. Ling Wei
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    Correspondence to Robert L. Flower.

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    Conflict of Interest

    Dr Darryl L. Irwin (Darryl.Irwin@agenabio.com) is the Senior Director, Applications Development at Agena Bioscience (formerly Sequenom Bioscience), Herston, Queensland, 4006, Australia. All other authors declare that they have no conflicts of interest relevant to the manuscript submitted to MOLECULAR BIOTECHNOLOGY.

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    Ling Wei and Genghis H. Lopez contributed equally to this study.

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    Wei, L., Lopez, G.H., Ji, Y. et al. Genotyping for Glycophorin GYP(B-A-B) Hybrid Genes Using a Single Nucleotide Polymorphism-Based Algorithm by Matrix-Assisted Laser Desorption/Ionisation, Time-of-Flight Mass Spectrometry. Mol Biotechnol 58, 665–671 (2016). https://doi.org/10.1007/s12033-016-9966-6

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    • DOI: https://doi.org/10.1007/s12033-016-9966-6

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