Sequence-Based Typing of HLA: An Improved Group-Specific Full-Length Gene Sequencing Approach

  • Christina E. M. Voorter
  • Fausto Palusci
  • Marcel G. J. Tilanus
Part of the Methods in Molecular Biology book series (MIMB, volume 1109)


Matching for HLA at the allele level is crucial for stem cell transplantation. The golden standard approach for allele definition of full gene polymorphism, the so-called high-resolution HLA typing, is sequence-based typing (SBT). Although the majority of the polymorphism for class I is located in exons 2 and 3 and for class II in exon 2, for allele definition it is necessary to unravel the complete coding and intron sequences leading to an ultrahigh HLA typing resolution at the allele level, i.e., a full-length gene polymorphism identification.

This chapter describes our recently developed SBT method for HLA-A, -B, -C, and -DQB1, that is based on full-length hemizygous Sanger sequencing of the alleles, separated by group-specific amplification using the low-resolution typing result as reference starting point. Group-specific amplification has already been established for DRB. This method enables a cost-efficient, user-friendly SBT approach resulting in a timely unambiguous HLA typing to an ultrahigh resolution level with minimal hands-on time.

Key words

Sequence-based typing Human leukocyte antigen HLA class I HLA class II Full-length HLA gene polymorphism Group-specific sequencing Unambiguous HLA typing 



The co-workers of the department are acknowledged for successfully introducing this validated SBT approach in routine diagnostics. The authors thank Diana van Bakel for her excellent secretarial assistance and Mathijs Groeneweg for the polymorphic index information.


  1. 1.
    Lee SJ, Klein J, Haagenson M et al (2007) High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood 110: 4576–4583PubMedCrossRefGoogle Scholar
  2. 2.
    Petersdorf EW (2008) Optimal HLA matching in hematopoietic cell transplantation. Curr Opin Immunol 20:588–593PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Crocchiolo R, Zino E, Vago L et al (2009) Nonpermissive HLA-DPB1 disparity is a significant independent risk factor for mortality after unrelated hematopoietic stem cell transplantation. Blood 114:1437–1444PubMedCrossRefGoogle Scholar
  4. 4.
    Fleischhauer K, Shaw BE, Gooley T et al (2012) Effect of T-cell-epitope matching at HLA-DPB1 in recipients of unrelated-donor haemopoietic-cell transplantation: a retrospective study. Lancet Oncol 13:366–374PubMedCrossRefGoogle Scholar
  5. 5.
    Hollenbach JA, Madbouly A, Gragert L et al (2012) A combined DPA1~DPB1 amino acid epitope is the primary unit of selection on the HLA-DP heterodimer. Immunogenetics 64:559–569Google Scholar
  6. 6.
    Spellman SR, Eapen M, Logan BR et al (2012) A perspective on the selection of unrelated donors and cord blood units for transplantation. Blood 120:259–265PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Amicosante M, Sanarico N, Berretta F et al (2001) Beryllium binding to HLA-DP molecule carrying the marker of susceptibility to berylliosis glutamate. Hum Immunol 62:686–693PubMedCrossRefGoogle Scholar
  8. 8.
    Billen EV, Christiaans MH, Doxiadis II et al (2010) HLA-DP antibodies before and after renal transplantation. Tissue Antigens 75:278–285PubMedCrossRefGoogle Scholar
  9. 9.
    Illing PT, Vivian JP, Dudek NL et al (2012) Immune self-reactivity triggered by drug-modified HLA-peptide repertoire. Nature 486:1–7Google Scholar
  10. 10.
    Sollid LM, Qiao SW, Anderson RP et al (2012) Nomenclature and listing of celiac disease relevant gluten T-cell epitopes restricted by HLA-DQ molecules. Immunogenetics 64: 455–460PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Smith L, Fidler S (2010) HLA typing technologies. In: Mehra N (ed) The HLA complex in biology and medicine. Jaypee Brothers Medical Publishers Ltd, New Delhi, pp 175–187Google Scholar
  12. 12.
    Voorter CEM, Meertens C, Palusci F et al (2012) Rare HLA alleles within the CWD groups redefined by a new SBT typing strategy. Hum Immunol 79:545Google Scholar
  13. 13.
    McGinnis MD, Conrad MP, Bouwens AGM et al (1995) Automated, solid-phase sequencing of DRB region genes using T7 sequencing chemistry and dye-labeled primers. Tissue Antigens 46:173–179PubMedCrossRefGoogle Scholar
  14. 14.
    Versluis LF, Rozemuller E, Tonks S et al (1993) High-resolution HLA-DPB typing based upon computerized analysis of data obtained by fluorescent sequencing of the amplified polymorphic exon 2. Hum Immunol 38:277–283PubMedCrossRefGoogle Scholar
  15. 15.
    Voorter CEM, Rozemuller EH, de Bruyn-Geraets D et al (1997) Comparison of DRB sequence-based typing using different strategies. Tissue Antigens 49:471–476PubMedCrossRefGoogle Scholar
  16. 16.
    Scheltinga SA, Johnston-Dow LA, White CB et al (1997) A generic sequencing based typing approach for the identification of HLA-A diversity. Hum Immunol 57:120–128PubMedCrossRefGoogle Scholar
  17. 17.
    Tilanus MGJ, Eliaou JF (1997) HLA sequencing based typing. In: Charron D (ed) Genetic diversity of HLA: functional and medical implications. EDK Press, Paris, pp 237–266Google Scholar
  18. 18.
    Voorter CEM, Kik MC, Van den Berg-Loonen EM (1998) High-resolution HLA typing for the DQB1 gene by sequence-based typing. Tissue Antigens 51:80–87PubMedCrossRefGoogle Scholar
  19. 19.
    van der Vlies SA, Voorter CEM, van den Berg-Loonen EM (1999) There is more to HLA-C than exons 2 and 3: sequencing of exons 1, 4 and 5. Tissue Antigens 54:169–177PubMedCrossRefGoogle Scholar
  20. 20.
    Swelsen WTN, Voorter CEM, van den Berg-Loonen EM (2002) Sequence analysis of exons 1, 2, 3, 4 and 5 of the HLA-B5/35 cross-reacting group. Tissue Antigens 60:224–234PubMedCrossRefGoogle Scholar
  21. 21.
    Swelsen WTN, Voorter CEM, van den Berg-Loonen EM (2004) Ambiguities of human leukocyte antigen-B resolved by sequence-based typing of exons 1, 4 and 5. Tissue Antigens 63:248–254PubMedCrossRefGoogle Scholar
  22. 22.
    Robinson J, Halliwell JA, McWilliam H et al (2013) The IMGT/HLA database. Nucleic Acids Res 41:D1222–D1227PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Reinders J, Rozemuller EH, Otten HG et al (2005) Identification of HLA-A*0111N: a synonymous substitution, introducing an alternative splice site in exon 3, silenced the expression of an HLA-A allele. Hum Immunol 66: 912–920PubMedCrossRefGoogle Scholar
  24. 24.
    Erlich H (2012) HLA DNA typing: past, present, and future. Tissue Antigens 80:1–11PubMedCrossRefGoogle Scholar
  25. 25.
    De Santis D, Dinauer D, Duke J et al (2013) 16(th) IHIW: review of HLA typing by NGS. Int J Immunogenet 40:72–76PubMedGoogle Scholar
  26. 26.
    Smith LK (2012) HLA typing by direct DNA sequencing. In: Christiansen F, Tait B (eds) Immunogenetics—methods and applications in clinical practice: methods in molecular biology. Humana, New York, pp 67–86Google Scholar
  27. 27.
    Bentley G, Higuchi R, Hoglund B et al (2009) High-resolution, high-throughput HLA genotyping by next-generation sequencing. Tissue Antigens 74:393–403PubMedCrossRefGoogle Scholar
  28. 28.
    Lind C, Ferriola D, Mackiewicz K et al (2010) Next-generation sequencing: the solution for high-resolution, unambiguous human leukocyte antigen typing. Hum Immunol 71: 1033–1042PubMedCrossRefGoogle Scholar
  29. 29.
    Parham P, Adams EJ, Arnett KL (1995) The origins of HLA-A, B, C polymorphism. Immunol Rev 143:141–180PubMedCrossRefGoogle Scholar
  30. 30.
    Dunn PP (2011) Human leucocyte antigen typing: techniques and technology, a critical appraisal. Int J Immunogenet 38:463–473PubMedCrossRefGoogle Scholar
  31. 31.
    Voorter CEM, de Bruyn-Geraets D, van den Berg-Loonen EM (1997) High-resolution HLA typing for the DRB3/4/5 genes by sequence-based typing. Tissue Antigens 50: 283–290PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Christina E. M. Voorter
    • 1
  • Fausto Palusci
    • 1
  • Marcel G. J. Tilanus
    • 1
  1. 1.Department of Transplantation Immunology, Tissue Typing LaboratoryMaastricht University Medical CenterMaastrichtThe Netherlands

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