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B-cell receptor sequencing of anti-citrullinated protein antibody (ACPA) IgG-expressing B cells indicates a selective advantage for the introduction of N-glycosylation sites during somatic hypermutation
  1. Rochelle D Vergroesen1,
  2. Linda M Slot1,
  3. Lise Hafkenscheid1,
  4. Marvyn T Koning2,
  5. Ellen I H van der Voort1,
  6. Christine A Grooff1,
  7. George Zervakis2,
  8. Hendrik Veelken2,
  9. Tom W J Huizinga1,
  10. Theo Rispens3,
  11. Hans U Scherer1,
  12. René E M Toes1
  1. 1 Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands
  2. 2 Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands
  3. 3 Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, Amsterdam, The Netherlands
  1. Correspondence to Dr Hans U Scherer, Department of Rheumatology, Leiden University Medical Center, P.O. Box 9600, Leiden 2300 RC, The Netherlands; H.U.Scherer{at}

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The majority of patients with rheumatoid arthritis (RA) harbours IgG antibodies targeting citrullinated protein antigens (ACPA). Recently, we showed that >90% of ACPA-IgG in serum are glycosylated in the variable domain.1 N-linked glycosylation requires a consensus sequence in the protein backbone (N-X-S/T (asparagine-X-serine/threonine), where X is any amino acid except proline), which is scarce in germline-encoded Ig variable region genes.2 3 Accordingly, hyperglycosylation of ACPA-IgG requires either clonal expansion of B cells expressing B-cell receptors (BCR) containing germline-encoded N-glycosylation sites or generation of de novo sites through somatic hypermutation (SHM).4

Here, we analysed the BCR repertoire of ACPA-expressing B cells to understand the molecular basis of this remarkable glycosylation. ACPA-expressing B cells were sorted as pools (10 cells per pool) from peripheral blood mononuclear cells of eight patients with ACPA-positive RA.5 Anchoring reverse transcription of immunoglobulin sequences and amplification by nested (ARTISAN) PCR-based BCR sequencing6 followed by full-length variable region IgG transcript analysis revealed high nucleotide mutation rates in 97 unique ACPA-IgG heavy chains (HC; mean ±SD: 52.86±16.73; figure 1A). 81% of these contained one or more N-glycosylation sites.

Figure 1

A high degree of somatic hypermutation in antibodies targeting citrullinated protein antigens (ACPA)-IgG clones which does not correlate with the frequency of N-glycosylation sites. Pool and single cells were sorted as described.5 All independent clones are defined as identical V, D, J genes and complementarity-determining region 3. (A) Immunoglobulin heavy variable region (IGHV) …

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  • RDV, LMS, LH and MTK contributed equally.

  • Contributors RDV, LMS, LH, TWJH, HUS and REMT designed the research. RDV, LMS, LH, MTK, CAG, EIHvdV and GZ performed the experiments. RDV, LMS, LH, MTK and TR analysed the data. RDV, LMS, LH, HUS and REMT wrote the manuscript. All the authors equally contributed to editing the manuscript.

  • Competing interests None declared.

  • Ethics approval Institutional Ethical Review Board.

  • Provenance and peer review Not commissioned; externally peer reviewed.