Gene conversion in human rearranged immunoglobulin genes.

Abstract

Over the past 20 years, many DNA sequences have been published suggesting that all or part of the V(H) segment of a rearranged immunoglobulin gene may be replaced in vivo. Two different mechanisms appear to be operating. One of these is very similar to primary V(D)J recombination, involving the RAG proteins acting upon recombination signal sequences, and this has recently been proven to occur. Other sequences, many of which show partial V(H) replacements with no addition of untemplated nucleotides at the V(H)-V(H) joint, have been proposed to occur by an unusual RAG-mediated recombination with the formation of hybrid (coding-to-signal) joints. These appear to occur in cells already undergoing somatic hypermutation in which, some authors are convinced, RAG genes are silenced. We recently proposed that the latter type of V(H) replacement might occur by homologous recombination initiated by the activity of AID (activation-induced cytidine deaminase), which is essential for somatic hypermutation and gene conversion. The latter has been observed in other species, but not in human Ig genes, so far. In this paper, we present a new analysis of sequences published as examples of the second type of rearrangement. This not only shows that AID recognition motifs occur in recombination regions but also that some sequences show replacement of central sections by a sequence from another gene, similar to gene conversion in the immunoglobulin genes of other species. These observations support the proposal that this type of rearrangement is likely to be AID-mediated rather than RAG-mediated and is consistent with gene conversion.

Full text

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Darlow, J.M. and Stott, D.I. (2006) Gene conversion in human rearranged
immunoglobulin genes. Immunogenetics 58(7):pp. 511-522.

http://eprints.gla.ac.uk/3646/

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John M. Darlow · David I. Stott

Gene conversion in human rearranged
immunoglobulin genes

J. M. Darlow (?) · D. I. Stott
Department of Immunology
Level 4
Glasgow Biomedical Research Centre
120 University Place
Glasgow, G12 8TA, UK
e-mail: john.m.darlow@clinmed.gla.ac.uk
Tel: +141 330 8417
Fax: +141 330 4297

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Abstract Over the past 20 years, many DNA sequences have been published
suggesting that all or part of the VH segment of a rearranged immunoglobulin
gene may be replaced in vivo. Two different mechanisms appear to be operating.
One of these is very similar to primary V(D)J recombination, involving the RAG
proteins acting upon recombination signal sequences, and this has recently been
proven to occur. Other sequences, many of which show partial VH replacements
with no addition of untemplated nucleotides at the VH-VH joint, have been
proposed to occur by an unusual RAG-mediated recombination with the formation
of hybrid (coding-to-signal) joints. These appear to occur in cells already
undergoing somatic hypermutation in which some authors are convinced that
RAG genes are silenced. We recently proposed that the latter type of VH
replacement might occur by homologous recombination initiated by the activity of
AID (activation-induced cytidine deaminase) which is essential for somatic
hypermutation and gene conversion. The latter has been observed in other species
but not so far in human Ig genes. Here we present a new analysis of sequences
published as examples of the second type of rearrangement. This not only shows
that AID recognition motifs occur in recombination regions but also that some
sequences show replacement of central sections by a sequence from another gene,
similar to gene conversion in the immunoglobulin genes of other species. These
observations support the proposal that this type of rearrangement is likely to be
AID- rather then RAG-mediated and is consistent with gene conversion.


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Key words Gene conversion - Genes, Immunoglobulin - Humans - Antibody
Diversity

Introduction

Evidence that the VH segment of a rearranged immunoglobulin VHDJH exon could
be replaced in vivo by a secondary rearrangement was first presented in 1986
(Kleinfield et al. 1986; Reth et al. 1986), since which time many more examples
have followed. The mechanism, recently conclusively demonstrated (Zhang et al.
2003), is similar to primary V(D)J recombination. It is mediated by the interaction
of the RAG protein complex on the recombination signal sequence (RSS) of the
incoming VH and a cryptic RSS, in the opposite direction, within the 3' end of the
rearranged VH, which acts as a surrogate for the RSS lost from the 5' end of the D
segment in the primary rearrangement. As in primary rearrangement, the RSS are
cleaved off and there is a variable amount of nucleotide deletion and addition at
the joined coding ends.
In 2000, evidence was presented for another type of VH replacement (Itoh et
al. 2000; Wilson et al. 2000), and a few more examples of this have followed. It
looks rather different from the first type, resembling homologous recombination
between the primarily rearranged VH gene segment and a new VH. As the
exchange usually occurs in a region in which both segments have the same
sequence, the exact point of recombination cannot be determined. It was proposed
(Itoh et al. 2000; Wilson et al. 2000) that it occurs at cryptic RSS but, if so, the

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recombination process would be quite different from normal V(D)J rearrangement
because the sequences proposed to function as cryptic RSS are in the same
orientation, one of them remains in the recombined product, there is no addition
of nucleotides at the point of exchange, and [with a single exception (Wilson et al.
2000)] no nucleotides are deleted from the gene sequence. Another problem with
this hypothesis is that most of the published examples show somatic
hypermutation (SHM) and the evidence points to some of this having occurred
before the secondary recombination. Thus the proposal that RAG-mediated
recombination is responsible goes against evidence that RAG expression has
ceased by the time that hypermutation begins. Finally, the sequences were
amplified from DNA isolated from pools of lymphocytes, giving the possibility
that the sequences could have arisen by hybridisation during PCR amplification.
In a recent review (Darlow and Stott 2005), we concluded that though hybrids
undoubtedly occur as an artefact during PCR, and may account for some of the
published examples of the second type of VH exchange (which we called Type 2
VH replacement), some were much less likely to be due to PCR hybridisation.
Firstly, two sequences of Itoh et al. (Itoh et al. 2000) obtained from a PCR using
VH Family 1 primers had a Family 1 segment hybridised to an original
rearrangement with a Family 3 VH segment. Their VH primer was specific for
Family 1 and they did not amplify any rearranged genes that started with a Family
3 VH segment. (These sequences are illustrated in our Fig. 3). Secondly, 23/26
related sequences from three independent sets of PCRs from a tumour
investigated by Lenze et al. (Lenze et al. 2003) were VH3-23(5')/VH3-07(3')

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