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

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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2

References

  1. 1.

    Daniels, G. (2013). Human blood groups. Oxford: Wiley-Blackwell Publishing.

    Google Scholar 

  2. 2.

    International Society of Blood Transfusion (ISBT), Red Cell Immunogenetics and Blood Group Terminology, Table of blood group antigens v5._151222.. [http://www.isbtweb.org/fileadmin/user_upload/Working_parties/WP_on_Red_Cell_Immunogenetics_and/Updates/Table_of_blood_group_antigens_within_systems_v5_151222.pdf].

  3. 3.

    Reid, M. E., Lomas-Francis, C., & Olsson, M. L. (2012). The blood group antigen facts book. San Diego: Elsevier Academic Press.

    Google Scholar 

  4. 4.

    Huang, C. H., & Blumenfeld, O. O. (1991). Multiple origins of the human glycophorin Sta gene. Identification of hot spots for independent unequal homologous recombinations. Journal of Biological Chemistry, 266, 23306–23314.

    CAS  Google Scholar 

  5. 5.

    Huang, C. H., Kikuchi, M., McCreary, J., & Blumenfeld, O. O. (1992). Gene conversion confined to a direct repeat of the acceptor splice site generates allelic diversity at human glycophorin (GYP) locus. Journal of Biological Chemistry, 267, 3336–3342.

    CAS  Google Scholar 

  6. 6.

    Huang, C. H., Skov, F., Daniels, G., Tippett, P., & Blumenfeld, O. O. (1992). Molecular analysis of human glycophorin MiIX gene shows a silent segment transfer and untemplated mutation resulting from gene conversion via sequence repeats. Blood, 80, 2379–2387.

    CAS  Google Scholar 

  7. 7.

    Huang, C. H., & Blumenfeld, O. O. (1991). Molecular genetics of human erythrocyte MiIII and MiVI glycophorins. Use of a pseudoexon in construction of two delta-alpha-delta hybrid genes resulting in antigenic diversification. Journal of Biological Chemistry, 266, 7248–7255.

    CAS  Google Scholar 

  8. 8.

    Storry, J. R., Poole, J., Condon, J., & Reid, M. E. (2000). Identification of a novel hybrid glycophorin gene encoding GP.Hop. Transfusion, 40, 560–565.

    CAS  Article  Google Scholar 

  9. 9.

    Green, C., Poole, J., Ford, D., & Glameyer, T. (1992). A postulated glycophorin B-A-B hybrid demonstrating heterogeneity of anti-Hop and anti-Nob sera. Transfusion Medicine (Oxford, England), 2, 67. P64 Abstract.

    Google Scholar 

  10. 10.

    Flower, R. L., Wei, L., Ji, Y. L., Luo, G. P., Lopez, G. H., & Hyland, C. A. (2013). GP.Kipp and GP.Yak BAB hybrid glycophorins: No difference in sequence or serology. Vox Sanguinis, 105, 10. 2A-S02-02 Abstract.

    Google Scholar 

  11. 11.

    Lopez, G. H., Wei, L., Ji, Y., Condon, J. A., Luo, G., Hyland, C. A., et al. (2016). GYP*Kip, a novel GYP(B-A-B) hybrid allele, encoding the MNS48 (KIPP) antigen. Transfusion, 56, 539–541.

    Article  Google Scholar 

  12. 12.

    Broadberry, R. E., & Lin, M. (1996). The distribution of the MiIII (Gp.Mur) phenotype among the population of Taiwan. Transfusion Medicine, 6, 145–148.

    CAS  Article  Google Scholar 

  13. 13.

    Poole, J., King, M. J., Mak, K. H., Liew, Y. W., Leong, S., & Chua, K. M. (1991). The MiIII phenotype among Chinese donors in Hong Kong: immunochemical and serological studies. Transfusion Medicine, 1, 169–175.

    CAS  Article  Google Scholar 

  14. 14.

    Wei, L., Shan, Z. G., Flower, R. L., Wang, Z., Wen, J. Z., Luo, G. P., et al. (2016). The distribution of MNS hybrid glycophorins with Mur antigen expression in Chinese donors including identification of a novel GYP.Bun allele. Vox Sanguinis,. doi:10.1111/vox.12421.

    Google Scholar 

  15. 15.

    Chandanyingyong, D., & Pejrachandra, S. (1975). Studies on the Miltenberger complex frequency in Thailand and family studies. Vox Sanguinis, 28, 152–155.

    CAS  Article  Google Scholar 

  16. 16.

    Palacajornsuk, P., Nathalang, O., Tantimavanich, S., Bejrachandra, S., & Reid, M. E. (2007). Detection of MNS hybrid molecules in the Thai population using PCR-SSP technique. Transfusion Medicine, 17, 169–174.

    CAS  Article  Google Scholar 

  17. 17.

    Uchikawa, M., Ogasawara, K., Suzuki, Y., Saito, M., Tsuneyama, H., Morimoto, K., et al. (2012). A new GP(B-A-B) hybrid molecule (GP.Yak) with Miltenberger phenotype. Vox Sanguinis, 103(Suppl. 1), 214. P-464 Abstract.

    Google Scholar 

  18. 18.

    Heathcote, D. J., Carroll, T. E., & Flower, R. L. (2011). Sixty years of antibodies to MNS system hybrid glycophorins: What have we learned? Transfusion Medicine Reviews, 25, 111–124.

    Article  Google Scholar 

  19. 19.

    Gassner, C., Meyer, S., Frey, B. M., & Vollmert, C. (2013). Matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry-based blood group genotyping–the alternative approach. Transfusion Medicine Reviews, 27, 2–9.

    Article  Google Scholar 

  20. 20.

    Haer-Wigman, L., Ji, Y., Loden, M., de Haas, M., van der Schoot, C. E., & Veldhuisen, B. (2013). Comprehensive genotyping for 18 blood group systems using a multiplex ligation-dependent probe amplification assay shows a high degree of accuracy. Transfusion, 53, 2899–2909.

    CAS  Article  Google Scholar 

  21. 21.

    Meyer, S., Vollmert, C., Trost, N., Bronnimann, C., Gottschalk, J., Buser, A., et al. (2014). High-throughput Kell, Kidd, and Duffy matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry-based blood group genotyping of 4000 donors shows close to full concordance with serotyping and detects new alleles. Transfusion, 54, 3198–3207.

    CAS  Article  Google Scholar 

  22. 22.

    Lopez, G. H., McBean, R. S., Wilson, B., Irwin, D. L., Liew, Y. W., Hyland, C. A., et al. (2015). Molecular typing for the Indian blood group associated 252G> C single nucleotide polymorphism in a selected cohort of Australian blood donors. Blood Transfusion, 13, 78–85.

    Google Scholar 

  23. 23.

    Meyer, S., Vollmert, C., Trost, N., Sigurdardottir, S., Portmann, C., Gottschalk, J., et al. (2016). MNSs genotyping by MALDI-TOF MS shows high concordance with serology, allows gene copy number testing and reveals new St(a) alleles. British Journal of Haematology. doi:10.1111/bjh.14095.

    Google Scholar 

  24. 24.

    Madden, H. J., Cleghorn, T. E., Allen, F. H, Jr., Rosenfield, R. E., & Mackeprang, M. (1964). A note on the relatively high frequency of St-a on the red blood cells of orientals, and report of a third example of anti-St-A. Vox Sanguinis, 9, 502–504.

    CAS  Article  Google Scholar 

  25. 25.

    Broadberry, R. E., Chang, F. C., Jan, Y. S., & Lin, M. (1998). The distribution of the red-cell Sta (Stones) antigen among the population of Taiwan. Transfusion Medicine, 8, 57–58.

    CAS  Article  Google Scholar 

Download references

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).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Robert L. Flower.

Ethics declarations

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.

Additional information

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

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 755 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Keywords

  • GP(B-A-B) hybrid glycophorin
  • GYP(B-A-B) genotyping
  • MALDI-TOF MS genotyping