Somatic immunoglobulin hypermutation

Immunoglobulin hypermutation provides the structural correlate for the affinity maturation of the antibody response. Characteristic modalities of this mechanism include a preponderance of point-mutations with prevalence of transitions over transversions, and the mutational hotspot RGYW sequence. Recent evidence suggests a mechanism whereby DNA-breaks induce error-prone DNA synthesis in immunoglobulin V(D)J regions by error-prone DNA polymerases. The nature of the targeting mechanism and the trans-factors effecting such breaks and their repair remain to be determined.     

Somatic hypermutation of immunoglobulin genes in human neonates

The antibody response in the young infant is limited in several ways; in particular, responses generally are of low affinity and restricted to IgM. This raises the question whether the affinity maturation process, consisting of somatic mutation of immunoglobulin genes coupled with selection of high-affinity variants, is operative in the neonate. Re-arranged V_H6 genes were amplified by polymerase chain reaction (PCR) from cord blood and from peripheral blood of infants. Heteroduplex analysis detected mutation in only 2/18 cord blood samples, while mutations were seen from about 10 days of age onwards. Cloning and sequencing of mutated neonatal V_H6 genes showed that mutated sequences contained relatively few mutations (one to three mutations per sequence) compared with published values of about 10 in adult IgM sequences. Selection was not evident in the majority of neonatal samples. Thus mutation can occur in the human neonate, but is minimal and generally not accompanied by selection. The age at which affinity maturation develops effectively is yet to be defined.     

Somatic hypermutation targeting is influenced by location within the immunoglobulin V region

The observed mutation pattern in immunoglobulin (Ig) V genes from peripheral B cells is influenced by several mechanisms, including the targeting of AID to specific DNA motifs, negative selection of B cells unable to express Ig receptor, and positive selection of B cells that carry affinity-increasing mutations. These influences, combined with biased codon usage, produce the well-known pattern of increased replacement mutation frequency in the CDR regions, and decreased replacement frequency in the framework regions. Through the analysis of over 12,000 mutated sequences, we show that the specific location in the V gene also significantly influences mutation accumulation. While this position-specific effect is partially explained by selection, it appears independently of the CDR/FWR structure. To further explore the specific targeting of SHM, we propose a statistical formalism describing the mutation probability of a sequence through the multiplication of independent probabilities. Using this model, we show that C→G (or G→C) mutations are almost as frequent as C→T and G→A mutations, in contrast with C→A (or G→T) mutations, which are as any other mutation. The proposed statistical framework allows us to precisely quantify the effect of V gene position, mutation substitution type, and micro-sequence specificity on the observed mutation pattern.     

Somatic hypermutation of immunoglobulin genes is independent of the Bloom's syndrome DNA helicase

Immunoglobulin gene somatic mutation leads to antibody affinity maturation through the introduction of multiple point mutations in the antigen binding site. No genes have as yet been identified that participate in this process. Bloom's syndrome (BS) is a chromosomal breakage disorder with a mutator phenotype. Most affected individuals exhibit an immunodeficiency of undetermined aetiology. The gene for this disorder, BLM, has recently been identified as a DNA helicase. If this gene were to play a role in immunoglobulin mutation, then people with BS may lack normally mutated antibodies. Since germ-line, non-mutated immunoglobulin genes generally produce low affinity antibodies, impaired helicase activity might be manifested as the immunodeficiency found in BS. Therefore, we asked whether BLM is specifically involved in immunoglobulin hypermutation. Sequences of immunoglobulin variable (V) regions were analysed from small unsorted blood samples obtained from BS individuals and compared with germ-line sequences. BS V regions displayed the normal distribution of mutations, indicating that the defect in BS is not related to the mechanism of somatic mutation. These data strongly argue against BLM being involved in this process. The genetic approach to identifying the genes involved in immunoglobulin mutation will require further studies of DNA repair- and immunodeficient individuals.     

Somatic hypermutation in antoimmune thyroid disease

Summary: Autoimmune thyroid disease is one of the most common autoimmune diseases. There is typically patient antibody (Ab) reactivity to one or more of the antigens thyroglobulin (Tg), thyroid peroxidase (TPO) and the thyroid stimulating hormone receptor (TSFlr). With the advent of combinatorial library technology, there has been an enormous increase in the number of sequences from Ab to Tg and TPO, The repertoire of both Tg and TPO Ab is restricted and indicates the importance of somatic hypermutation in the development of the high affinity, Ab response. However, there are still too few sequences to determine patterns in which the mutation occurs, which residues are introduced during substitution and how individual substitutions affect the affinity of the Ab, Ab to the TSHr are of far greater pathological significance than those to Tg and TPO, but the current repertoire of Ab to the TSHr has yet to include the high affinity IgG Ab characteristic of patient serum Ab, Instructive analysis of the role of somatic hypermutation in the development of TSHr Ab therefore still awaits the isolation of the pathologically active repertoire. Despite this, the Ab response in thyroid autoimmunity remains one of the best characterised of human autoimmune diseases.     

Somatic hypermutation of immunoglobulin variable region genes: focus on follicular lymphoma and multiple myeloma

Summary: Analysis of the rearranged immunoglobulin variable region gene hypermutation has provided important information concerning the clonal history and ontogenetic origin of various B-cell lymphoproliferative disorders. Under the selective pressure of antigen, mutational events in immunoglobulin genes will fine tune survival of B-cell clones bearing immunoglobulin with high affinity for antigen. Our studies aimed at analyzing neoplastic disorders originating from germinal and post-germinal center B-cells: follicular lymphoma and multiple myeloma. respectively. Despite the already acknowledged evidence for a selectable distribution of mutations within the clonal immunoglobulin variable heavy chain genes, very little is known about the contribution of light chains in the process of antigen selection. In follicular lymphoma. a more limited pattern of somatic mutation with less evidence of antigen selection was observed in variable K light chain genes (40%) than in their partner heavy chain genes (80%). In myeloma, hypermutation of variable light chain genes, with a distribution suggestive of antigen selection, was frequently observed. Based on these data and recent reports it appears that the light chain expressed by the clonogenic myeloma B-cells plays a pivotal role in the antigen selection process. Additionally, abortive K light chain variable region genes in X-expressing myeloma carried a significant number of somatic mutations indicating that the cell of origin is open to the hypermutation machinery at that particular developmental stage irrespective of antigen selection.     

Somatic hypermutation in mouse λ chains

Summary: The frequency and distribution of somatic hypermutation in immunoglobulin genes and the effect of amino acid substitution on the structure/function of antibodies were studied using hybridomas that secrete anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) monoclonal antibodies bearing Xl chains. A high frequency of mutation was observed in V-J exons and J-C introns of rearranged and active λ1 chains but not in the 5'-non-coding regions of these chains. Since a similar distribution was observed in inactive λ2 chain genes, 5'-non-coding regions containing a promoter were considered to be protected from mutation in view of their apparent importance. Using transgenic mice carrying chloramphenicol acetyl transferase transgenes driven by the VH promoter and heavy-chain intron enhancer, it was also revealed that these cis-acting elements are important in the induction of somatic hypermutation and are capable of inducing mutation even in non-immunoglobulin genes.     

Somatic hypermutation and mismatch repair in non-B cells

Mismatch repair contributes to hypermutation in B lymphocytes, both by increasing the frequency of mutations and by changing the mutational patterns. In this paper, we investigated whether or not mismatch repair influences activation-induced cytidine deaminase (AID)-mediated hypermutation in a non-B lymphocyte line. We did so by regulating expression of MutL homologue MLH 1, which is essential in mismatch repair, in a kidney cell line that had been transduced by an AID-containing vector. Whether or not MLH1 was expressed, we found no difference in the mutation rates of an indicator gene. We conclude that in order to contribute to hypermutation, mismatch repair needs additional factors that are present in activated B lymphocytes, but absent in the cell line investigated.     

Somatic hypermutation of human immunoglobulin heavy chain genes: targeting of RGYW motifs on both DNA strands

The impact of the somatic hypermutational machinery was examined by analyzing the frequency and distribution of mutations in nonproductive V_HDJ_H rearrangements obtained from individual human peripheral B cells. A strong bias toward nucleotide substitutions within the quadruplet motif RGYW was observed. In addition, there was a comparably increased frequency of mutations of the inverse repeat of RGYW, WRCY. Together, mutations of RGYW / WRCY accounted for 37 % of all nucleotide substitutions. No significant strand polarity of the distribution of mutations was evident when nucleotide substitutions of highly mutated quartets and triplets as well as of their inverse repeats were analyzed. Furthermore, detailed analysis of mutations of specific triplets, such as AGC and TAC provided evidence that they were mutated more frequently when they were included within RGYW and WRCY, respectively. Despite being a target of the mutational machinery, neither RGYW nor WRCY was mutated in the absence of a large number of substitutions of other nucleotides in the same sequence. These results indicate that the mutational machinery targets RGYW sequences for mutations on either DNA strand and do not support the contention that the mutational machinery exhibits DNA strand polarity.     

Somatic hypermutation in normal and transformed human B cells

Summary: In the human, most IgM⁺IgD⁺ as well as CD5* peripheral blood B cells express unmutated V genes and thus can be assigned to a pre-germinal centre (GC) stage of development. The memory B-cell compartment generated in die GC reaction and characterized by cells bearing somatically mutated V-region genes consists not only of class-switched cells, but also of lgM-only B cells and perhaps a subset of IgM⁺IgD⁺ B cells expressing the CD27 antigen. Comparison of the rearranged V-region genes of human B-cell lymphomas with those of the normal B-cell subsets allows the identification of the progenitor cells of these tumours in terms of their stage of maturation. On this basis, most B-cell on-Hodgkin lymphomas, and in addition Hodgkin and Reed-Stern berg (HRS) cells in Hodgkin's disease (HD). are derived from B cells ac a GC or post-GC stage of development. The mutation pattern indicates that the precursors of the tumour clones have been stringently selected for expression of a functional antigen receptor with one notable exception: HRS cells in classical (but: not lymphocyte-predominant) HD appear to be derived from "crippled" GC B cells. Sequence analysis of rearranged V genes amplified from single tonsillar GC B cells revealed that the somatic hypermutation process introduces deletions and/or insertions into V-region genes more frequently that indicated by previous investigations. Presumably, this feature of the hypermutation mechanism is often responsible for the generation of heavy chain disease, and also several types of chromosomal translocations of oncogenes into immunoglobulin loci in human B-cell lymphomas.     

Controlling somatic hypermutation in immunoglobulin variable and switch regions

Activation-induced deaminase (AID) is a B-cell-specific enzyme required for initiating the mechanisms of affinity maturation and isotype switching of antibodies. AID functions by deaminating cytosine to uracil in DNA, which initiates a cascade of events resulting in mutations and strand breaks in the immunoglobulin loci. There is an intricate interplay between faithful DNA repair and mutagenic DNA repair during somatic hypermutation, in that some proteins from accurate repair pathways are also involved in mutagenesis. One factor that shifts the balance from faithful to mutagenic repair is the genomic sequence of the switch regions. Indeed, the sequence of the switch μ region is designed to maximize AID access to increase the abundance of clustered dU bases. The frequency and proximity of these dU nucleotides then in turn inhibit faithful repair and promote strand breaks.     

Somatic hypermutation and diverse immunoglobulin gene usage in the human antibody response to the capsular polysaccharide of Streptococcus pneumoniae Type 6B

Combinatorial cloning and expression library analysis were used to determine the expressed human antibody repertoire specific for the capsular polysaccharide (PS) of Streptococcus pneumoniae serotype 6B. Sequence analysis of 55 6B-specific antibody Fab fragments isolated from six vaccinated donors reveal that different individuals used a variety of heavy and light chain germ line variable (V) region genes to form pneumococcal capsular PS (PPS) 6B-specific paratopes. Within each donor, however, the response was more restricted, with five of the six donors using at most one or two gene pairs to form combining sites. Analysis also indicated that although the response in each donor was oligoclonal in terms of variable gene usage, the combination of extensive somatic hypermutation, deletion of germ line-encoded residues, insertion of non-germ line-encoded residues, and intraclonal isotype switching generated a surprising degree of paratope diversity within the individuals analyzed. In contrast to previously studied PS-specific responses, we find that the PPS 6B repertoire makes use of a diverse collection of heavy-chain and light-chain V region gene products to form specific paratopes, with no apparent tendency for conservation of immunoglobulin gene usage between individuals.     

Targets of somatic hypermutation within immunoglobulin light chain genes in zebrafish

In mammals, somatic hypermutation (SHM) of immunoglobulin (Ig) genes is critical for the generation of high‐affinity antibodies and effective immune responses. Knowledge of sequence‐specific biases in the targeting of somatic mutations can be useful for studies aimed at understanding antibody repertoires produced in response to infections, B‐cell neoplasms, or autoimmune disease. To evaluate potential nucleotide targets of somatic mutation in zebrafish (Danio rerio), an enriched IgL cDNA library was constructed and > 250 randomly selected clones were sequenced and analysed. In total, 55 unique VJ‐C sequences were identified encoding a total of 125 mutations. Mutations were most prevalent in V_L with a bias towards single base transitions and increased mutation in the complementarity‐determining regions (CDRs). Overall, mutations were overrepresented at WRC H/DG YW motifs suggestive of activation‐induced cytidine deaminase (AID) targeting which is common in mice and humans. In contrast to mammalian models, N and P addition was not observed and mutations at AID hotspots were largely restricted to palindromic WRC H/DG YW motifs. Mutability indexes for di‐ and trinucleotide combinations confirmed C /G targets within WRC H/DG YW motifs to be statistically significant mutational hotspots and showed trinucleotides ATC and ATG to be mutation coldspots. Additive mutations in VJ‐C sequences revealed patterns of clonal expansion consistent with affinity maturation responses seen in higher vertebrates. Taken together, the data reveal specific nucleotide targets of SHM in zebrafish and suggest that AID and affinity maturation contribute to antibody diversification in this emerging immunological model.     

Normal immunoglobulin gene somatic hypermutation in Pol kappa-Pol iota double-deficient mice

Somatic hypermutation (SHM) occurs in the variable region of immunoglobulin genes in germinal center B cells where it plays an important role in affinity maturation of the T cell-dependent immune response. Although the precise mechanism of SHM is still unknown, it has been suggested that error-prone DNA polymerases (Pol) are involved in SHM. Poliota is a member of the error-prone Y-family of DNA polymerases which exhibit translesion synthesis activity in vitro and are highly mutagenic when replicating on non-damaged DNA templates. In BL2 cell line stimulated to induce SHM, the induction is Poliota-dependent. However, in 129-derived strains of mice deficient in Poliota, SHM is normal. One possible explanation for this discrepancy is that a Poliota deficiency in mice might be compensated for by another error-prone DNA polymerase, such as Polkappa, which also belongs to the Y-family of DNA polymerases. Although SHM in Polkappa-deficient mice is normal, their deficiency might be compensated for by Poliota. In this study, we generated Polkappa-Poliota double-deficient mice and examined them for SHM. We found that the double-deficient mice had the normal SHM frequency and profile, rendering them indistinguishable from Polkappa-deficient mice and thus conclude that Poliota and Polkappa are dispensable for SHM in mice.     

Somatic hypermutation and antigen-driven selection of B cells are altered in autoimmune diseases

B cells have been found to play a critical role in the pathogenesis of several autoimmune (AI) diseases. A common feature amongst many AI diseases is the formation of ectopic germinal centers (GC) within the afflicted tissue or organ, in which activated B cells expand and undergo somatic hypermutation (SHM) and antigen-driven selection on their immunoglobulin variable region (IgV) genes. However, it is not yet clear whether these processes occurring in ectopic GCs are identical to those in normal GCs. The analysis of IgV mutations has aided in revealing many aspects concerning B cell expansion, mutation and selection in GC reactions. We have applied several mutation analysis methods, based on lineage tree construction, to a large set of data, containing IgV productive and non-productive heavy and light chain sequences from several different tissues, to examine three of the most profoundly studied AI diseases – Rheumatoid Arthritis (RA), Multiple Sclerosis (MS) and Sjögren’s Syndrome (SS). We have found that RA and MS sequences exhibited normal mutation spectra and targeting motifs, but a stricter selection compared to normal controls, which was more apparent in RA. SS sequence analysis results deviated from normal controls in both mutation spectra and indications of selection, also showing differences between light and heavy chain IgV and between different tissues. The differences revealed between AI diseases and normal control mutation patterns may result from the different microenvironmental influences to which ectopic GCs are exposed, relative to those in normal secondary lymphoid tissues.