Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter to the Editor
  • Published:

Universal N-glycosylation sites introduced into the B-cell receptor of follicular lymphoma by somatic mutation: a second tumorigenic event?

Leukemia volume 20, pages 530–534 (2006)Cite this article

We have previously shown that variable region heavy chain (VH) genes in FL commonly acquire sequence motifs by somatic mutation that serve as sites for N-glycosylation.1 We further showed that subsets of aggressive B NHL acquire sites2 in contrast to sequences of myeloma, CLL and normal B cells where this is rare.1 These data suggest positive selection and a critical role for the introduced glycosylation sites in the pathogenesis of FL.

In all, 26 different N-glycosylation motifs were observed. The most commonly used was NIS, which was observed in nine (21%) of VH genes, but which was only seen once in VL. Other repeatedly used motifs included NIT (n=5), NSS, NTS, NYT and NLT (all n=4). The disfavoured motifs NDS, NLS and NWS were found in four FL and involved two VH genes and two VL genes, but in all cases additional sites were present in either the V gene itself or its partner chain. Motifs were created by a range of nucleotide changes. The codon for Asn (AAU or AAC) was generated by mutation rather than being derived from germline sequence in 41 of 45 (91%) instances. ‘Starter’ sequences for motif generation depended on the donor germline VH. For example, YIS present in V3-48 and V3-11 gene segments at CDR2 was mutated in six sequences to NIS and in two cases to NIT. Mutational changes of Ser to Asn were relatively common (12/45), likely reflecting the presence of a hot spot of SHM at codon 50 in CDR2 (see below). Other common amino-acid (aa) replacements in codon 1 affected Tyr (15/45) and Thr (6/45), where replacement mostly required one nucleotide change to generate the starter Asn. Acquisition of sites was indeed predominantly (73%) achieved by a single aa replacement to Asn at position 1 of the motif. At the nucleotide level, the first codon underwent mutations of one (68%), two (29%) and rarely three (2%) nucleotides. In a substantial proportion (24%), additional, non-essential aa replacements in codon 2 (n=4) or codon 3 (n=7) were found. It is interesting to note that unlike the Asn in codon 1, the C-terminal Ser of the motif was most commonly found either in the natural sequence (n=20) or mutated from Ser to Thr (n=6); 18 motifs therefore ended in Thr, of which only one-third were present in the germline sequence. A change of Thr to Ser was only observed once. Where codon 3 was mutated (42%), SHM had affected one (63%) or two (37%) nucleotide mutations. In all, 18% of sites were created through an essential aa replacement at both positions 1 and 3 of the motif, but only in a minority (9%) by an essential aa replacement at position 3 alone. A more detailed analysis of the generation of N-glycosylation sites in the VH CDR2 was performed and included sequences from cited references5, 6, 8 (Table 2). All 47 sequences with CDR2 motifs used VH3 (n=35) and VH4 (n=12) genes. Here, 60% had acquired a site at aa position 50–52. Codon 50 is a recognized hotspot for SHM in both productively and non-productively rearranged alleles of normal B cells and affected ∼20% of cases.10 A total of 60% of FL sequences showed a replacement mutation here (28/47). This may, in part, be explained by the relative over representation of the VH3 genes and in particular V3-48, for which the majority of sites were generated by aa replacement at position 50. In contrast, there is no recognized hotspot for SHM at codon 52 of VH,10 but we find that codon 52 is mutated in 21 of 34 cases. The high incidence of codon 52 changes in CDR2 motifs further point to selective pressure on FL to acquire N-glycosylation sites in their V genes. Eight further cases using the gene segments V3-11, V3-23 or V3-30 generated sites by a replacement mutation at codon 50, the majority (6/8) with an additional permissive mutation in codon 52. For segments V3-07, all sites were generated by the replacement of Lys to either Ser or Thr at position 52. For segments V3-15, all sites were generated by the replacement of Ser to Asn at position 52a. In the remaining gene segments, sites did not appear to focus to any one aa position. Strikingly in VL there is a complete absence of motifs in CDR2 in spite of a known hotspot for SHM in codon 52. This points to a selection against further acquisition of sites here, as in normal B cells CDR2 (and CDR1) appear to be preferentially subjected to SHM.11 Other common hotspots for SHM such as codons 31, 55 or 82a were not used to generate novel sites and we observed no repeated location of sites in VL. Overall analysis of the affected codons provides further evidence for positive selection. Additionally, the accumulation of motifs in CDR3 of FL compared to normal B cells provides new data that this region in the V genes is affected by SHM, which had previously been difficult to show directly in the absence of a germline counterpart.

Table 2 Analysis of somatic mutations giving rise to N-glycosylation motifs: a focus on VH CDR2
Full size table

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3

References

  1. Zhu D, McCarthy H, Ottensmeier CH, Johnson P, Hamblin TJ, Stevenson FK . Acquisition of potential N-glycosylation sites in the immunoglobulin variable region by somatic mutation is a distinctive feature of follicular lymphoma. Blood 2002; 99: 2562–2568.

    Article  CAS  PubMed  Google Scholar 

  2. Zhu D, Ottensmeier CH, Du MQ, McCarthy H, Stevenson FK . Incidence of potential glycosylation sites in immunoglobulin variable regions distinguishes between subsets of Burkitt’s lymphoma and mucosa-associated lymphoid tissue lymphoma. Br J Haematol 2003; 120: 217–222.

    Article  CAS  PubMed  Google Scholar 

  3. Bahler DW, Campbell MJ, Hart S, Miller RA, Levy S, Levy R . Ig VH gene expression among human follicular lymphomas. Blood 1991; 78: 1561–1568.

    CAS  PubMed  Google Scholar 

  4. Hsu FJ, Levy R . Preferential use of the VH4 Ig gene family by diffuse large-cell lymphoma. Blood 1995; 86: 3072–3082.

    CAS  PubMed  Google Scholar 

  5. Stamatopoulos K, Kosmas C, Papadaki T, Pouliou E, Belessi C, Afendaki S et al. Follicular lymphoma immunoglobulin kappa light chains are affected by the antigen selection process, but to a lesser degree than their partner heavy chains. Br J Haematol 1997; 96: 132–146.

    Article  CAS  PubMed  Google Scholar 

  6. Noppe SM, Heirman C, Bakkus MH, Brissinck J, Schots R, Thielemans K . The genetic variability of the VH genes in follicular lymphoma: the impact of the hypermutation mechanism. Br J Haematol 1999; 107: 625–640.

    Article  CAS  PubMed  Google Scholar 

  7. Aarts WM, Bende RJ, Steenbergen EJ, Kluin PM, Ooms EC, Pals ST et al. Variable heavy chain gene analysis of follicular lymphomas: correlation between heavy chain isotype expression and somatic mutation load. Blood 2000; 95: 2922–2929.

    CAS  PubMed  Google Scholar 

  8. Zabalegui N, de Cerio AL, Inoges S, Rodriguez-Calvillo M, Perez-Calvo J, Hernandez M et al. Acquired potential N-glycosylation sites within the tumor-specific immunoglobulin heavy chains of B-cell malignancies. Haematologica 2004; 89: 541–546.

    CAS  PubMed  Google Scholar 

  9. Kasturi L, Chen H, Shakin-Eshleman SH . Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors. Biochem J 1997; 323 (Part 2): 415–419.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Dorner T, Brezinschek HP, Brezinschek RI, Foster SJ, Domiati-Saad R, Lipsky PE . Analysis of the frequency and pattern of somatic mutations within nonproductively rearranged human variable heavy chain genes. J Immunol 1997; 158: 2779–2789.

    CAS  PubMed  Google Scholar 

  11. Foster SJ, Dorner T, Lipsky PE . Targeting and subsequent selection of somatic hypermutations in the human V kappa repertoire. Eur J Immunol 1999; 29: 3122–3132.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was funded by Cancer Research UK and the Nessex Cancer Trust.

Author information

Authors and Affiliations

  1. Cancer Sciences Division, Somers Cancer Sciences Building, University of Southampton, Southampton, UK

    K J McCann, P W M Johnson, F K Stevenson & C H Ottensmeier

Authors
  1. K J McCann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P W M Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. F K Stevenson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C H Ottensmeier
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C H Ottensmeier.

Rights and permissions

Reprints and permissions

About this article

Cite this article

McCann, K., Johnson, P., Stevenson, F. et al. Universal N-glycosylation sites introduced into the B-cell receptor of follicular lymphoma by somatic mutation: a second tumorigenic event?. Leukemia 20, 530–534 (2006). https://doi.org/10.1038/sj.leu.2404095

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.leu.2404095

This article is cited by

Search

Advanced search

Quick links