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Results for “somatic hypermutation hotspots”

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At least 19 recordsLinked to original sources

PubMed2006

Computational analyses show A-to-G mutations correlate with nascent mRNA hairpins at somatic hypermutation hotspots.

Activation-induced cytidine deaminase (AID) initiates Phase I somatic hypermutation (SHM) of antibody genes by deaminating deoxy-cytosine to deoxy-uracil (C-to-U). These lesions trigger Phase II, a poorly understood process of error-prone repair targeting A-T pairs by DNA polymerase eta (Pol eta). Since Pol eta is also a reverse transcriptase, Phase II could involve copying off RNA as well as DNA templates. We explore this idea further since in an RNA-based pathway it is conceivable that adenosine-to-inosine (A-to-I) RNA editing causes A-to-G transitions since I like G pairs with C. Adenosine deaminases (ADARs) are known to preferentially edit A nucleotides that are preceded by an A or U (W) in double-stranded RNA substrates. On this assumption and using a theoretical bioinformatics approach we show that a significant and specific correlation (P<0.002) exists between the frequency of WA-to-WG mutations and the number of mRNA hairpins that could potentially form at the mutation site. This implies roles for both RNA editing and reverse transcription during SHM in vivo and suggests definitive genetic experiments targeting the appropriate ADAR1 isoform (gammaINF-ADAR1) and/or Ig pre-mRNA templates.

Computational Biology↗

PubMed2001

The PU.1 and NF-EM5 binding motifs in the Igkappa 3' enhancer are responsible for directing somatic hypermutations to the intrinsic hotspots in the transgenic Vkappa gene.

Somatic hypermutation is a key mechanism in generating Ig with higher affinities to antigen, a process known as affinity maturation. Using Igkappa transgenes, the 3' enhancer (kappaE3') has been shown to play an important role in introducing hypermutations. In order to identify the cis-acting elements that regulate hypermutagenesis, we have generated transgenic substrates containing mutations/deletions in the kappaE3' region. Here, we report that base substitutions in the kappaE3', either in the PU.1 or in the NF-EM5 binding motif, not only reduce the mutation rate but also disrupt the directed mutagenesis in the intrinsic hotspots of the Igkappa transgene.

3' Untranslated Regions↗

PubMed2005

DNA polymerase theta contributes to the generation of C/G mutations during somatic hypermutation of Ig genes.

Somatic hypermutation of Ig variable region genes is initiated by activation-induced cytidine deaminase; however, the activity of multiple DNA polymerases is required to ultimately introduce mutations. DNA polymerase eta (Poleta) has been implicated in mutations at A/T, but polymerases involved in C/G mutations have not been identified. We have generated mutant mice expressing DNA polymerase (Pol) specifically devoid of polymerase activity. Compared with WT mice, Polq-inactive (Polq, the gene encoding Pol) mice exhibited a reduced level of serum IgM and IgG1. The mutant mice mounted relatively normal primary and secondary immune responses to a T-dependent antigen, but the production of high-affinity specific antibodies was partially impaired. Analysis of the J(H)4 intronic sequences revealed a slight reduction in the overall mutation frequency in Polq-inactive mice. Remarkably, although mutations at A/T were unaffected, mutations at C/G were significantly decreased, indicating an important, albeit not exclusive, role for Pol activity. The reduction of C/G mutations was particularly focused on the intrinsic somatic hypermutation hotspots and both transitions and transversions were similarly reduced. These findings, together with the recent observation that Pol efficiently catalyzes the bypass of abasic sites, lead us to propose that Pol introduces mutations at C/G by replicating over abasic sites generated via uracil-DNA glycosylase.

Animals↗

PubMed2004

Diversity and repertoire of IgW and IgM VH families in the newborn nurse shark.

BACKGROUND: Adult cartilaginous fish express three immunoglobulin (Ig) isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric) IgM and monomeric/dimeric IgM1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric) IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3) with non-templated nucleotide (N-region) addition, which suggests that, unlike in many other vertebrates, terminal deoxynucleotidyl transferase (TdT) expressed at birth is functional. IgW is present in the lungfish, a bony fish sharing a common ancestor with sharks 460 million years ago, implying that the IgW VH family is as old as the IgM VH family. This nurse shark study examined the IgM and IgW VH repertoire from birth through adult life, and analyzed the phylogenetic relationships of these gene families. RESULTS: IgM and IgW VH cDNA clones isolated from newborn nurse shark primary and secondary lymphoid tissues had highly diverse and unique CDR3 with N-region addition and VDJ gene rearrangement, implicating functional TdT and RAG gene activity. Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults. The IgM clones are all included in a conventional VH family that can be classified into five discrete groups, none of which is orthologous to IgM VH genes in other elasmobranchs. In addition, a novel divergent VH family was orthologous to a published monotypic VH horn shark family. IgW VH genes have diverged sufficiently to form three families. IgM and IgW VH serine codons using the potential somatic hypermutation hotspot sequence occur mainly in VH framework 1 (FR1) and CDR1. Phylogenetic analysis of cartilaginous fish and lungfish IgM and IgW demonstrated they form two major ancient gene groups; furthermore, these VH genes generally diversify (duplicate and diverge) within a species. CONCLUSION: As in ratfish, sandbar and horn sharks, most nurse shark IgM VH genes are from one family with multiple, heterogeneous loci. Their IgW VH genes have diversified, forming at least three families. The neonatal shark Ig VH CDR3 repertoire, diversified via N-region addition, is shorter than the adult VDJ junction, suggesting one means of postnatal repertoire diversification is expression of longer CDR3 junctions.

Age Factors↗

PubMed2005

Natural evolution of a human virus-specific antibody gene repertoire by somatic hypermutation requires both hotspot-directed and randomly-directed processes.

Somatic hypermutation of antibody genes is mediated by activation-induced cytidine deaminase and targets primarily hotspot motifs. We tested the hypothesis that the antibody variable genes of virus-specific B cells from infants exhibit a decreased frequency of somatic mutations compared with adults. We also sought to determine whether virus-specific B cells exhibit predominantly hotspot or randomly directed processes. We analyzed somatic mutations in rotavirus (RV)-specific B cells from otherwise healthy but recently RV-infected infants or adults in comparison with B cells from healthy volunteers not recently infected. We compared these antibody variable gene sequences with those derived from RV-specific B cells from an adult patient with X-linked hyper-IgM syndrome (XHIM). We found that the overall mutational frequency within the antibody variable region was lowest in RV-specific B cells from RV-infected infants, followed by randomly selected B cells, followed by RV-specific B cells from the patient with XHIM. RV-specific memory B cells from healthy adults exhibited the highest frequency of mutations. Approximately half of mutations in random or RV-specific B cells from adults or infants occurred at the DGYW/WRCH or WA/TW hotspot motifs. These findings suggest that virus-specific antibodies require both hotspot and randomly-directed processes.

Adult↗

PubMed2001

Somatic hypermutation targeting to intrinsic hotspots of immunoglobulin genes in follicular lymphoma and multiple myeloma.

In this study, we analyzed the targeting of the somatic hypermutation (SHM) mechanism at specific hotspot sequence motifs in the V(H) and Vkappa genes of 10 follicular lymphoma (FL) cases and the Vkappa and Vlambda genes of 11 kappa- and six lamda-light chain expressing multiple myeloma (MM) cases. These sequences were analyzed for targeting of specific motifs, ie certain highly mutable trinucleotides (3-NTPs), the tetranucleotide (4-NTP) RGYW and its complementary, WRCY (where R = purine, Y = pyrimidine and W = A or T). Comparisons were carried out between mutation frequencies in RGYW vs WRCY and the incidence of mutations in complementarity determining region (CDR)-1 vs CDR2 vs CDR3. Statistically significant differences were obtained when comparing: (1) the ratio of mutations in 4-NTPs (RGYW, WRCY, RGYW+WRCY)/mutations in the whole V sequence in MM-Vkappa vs MM-Vlamda; (2) the total number of mutated 4-NTPs in MM-Vkappa vs FL-Vkappa; (3) the number of mutated RGYW 4-NTPs in MM-Vkappa vs FL-Vkappa and FL-V(H) vs FL-Vkappa; (4) the number of mutated WRCY 4-NTPs in MM-Vkappa vs FL-Vkappa (P= 0.006) and FL-V(H) vs FL-Vkappa; (5) the targeting of RGYW vs WRCY in the CDRs of FL-V(H) genes. Similar results (regarding statistical significance) were obtained when undertaking intergroup comparisons for 3-NTPs. These findings conform well with relevant data derived from normal peripheral B cells. The differences observed in favor of 4-NTP (RGYW and WRCY) targeting in FL-V(H) vs FL-Vkappa and MM-Vkappa vs FL-Vkappa may implicate differences in the evolution of SHM coupled with selection in different stages of B cell ontogeny. Several explanations can be offered for the fact that hotspot sequences were not always targeted by SHM in FL and MM: (1) other unrecognized motifs may be targets of SHM; (2) 'inappropriately' introduced mutations were fixed and propagated by the neoplastic process; (3) certain FL and MM cases might have lost their ability to correct mutations introduced in classic hotspots due to deficient mismatch-repair (MMR) mechanisms; conversely, in other cases with intact MMR function, the hotspot to non-hotspot targeting of somatic hypermutation is balanced.

Base Sequence↗

PubMed1998

Somatic hypermutation introduces insertions and deletions into immunoglobulin V genes.

During a germinal center reaction, random mutations are introduced into immunoglobulin V genes to increase the affinity of antibody molecules and to further diversify the B cell repertoire. Antigen-directed selection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response. The mutations of Ig genes are typically basepair substitutions, although DNA insertions and deletions have been reported to occur at a low frequency. In this study, we describe five insertion and four deletion events in otherwise somatically mutated VH gene cDNA molecules. Two of these insertions and all four deletions were obtained through the sequencing of 395 cDNA clones (approximately 110,000 nucleotides) from CD38+IgD- germinal center, and CD38-IgD- memory B cell populations from a single human tonsil. No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic VH4 repertoire of the tonsil donor. These six insertions or deletions and three additional insertion events isolated from other sources occurred as triplets or multiples thereof, leaving the transcripts in frame. Additionally, 8 of 9 of these events occurred in the CDR1 or CDR2, following a pattern consistent with selection, and making it unlikely that these events were artifacts of the experimental system. The lack of similar instances in unmutated IgD+CD38- follicular mantle cDNA clones statistically associates these events to the somatic hypermutation process (P = 0.014). Close scrutiny of the 9 insertion/deletion events reported here, and of 25 additional insertions or deletions collected from the literature, suggest that secondary structural elements in the DNA sequences capable of producing loop intermediates may be a prerequisite in most instances. Furthermore, these events most frequently involve sequence motifs resembling known intrinsic hotspots of somatic hypermutation. These insertion/deletion events are consistent with models of somatic hypermutation involving an unstable polymerase enzyme complex lacking proofreading capabilities, and suggest a downregulation or alteration of DNA repair at the V locus during the hypermutation process.

Amino Acid Sequence↗

PubMed2026

Genome-wide etiology analysis of autoimmune hypothyroidism supports somatic mutations of at-risk DNA as the underlying cause.

Autoimmune hypothyroidism (AIHT) is the most common autoimmune disease. Through an unidentified mechanism, the immune system attacks the thyroid gland, destroys thyroid follicular cells, and causes hypothyroidism. A new theory poses that all DNA is continuously damaged and, as a result, is exposed to somatic mutations at a constant rate. Based on this theory, several assumptions related to epidemiology and DNA sequence can be made. These have been summarized as a method called genome-wide etiology analysis (GWEA) to facilitate the interpretation of GWAS results of autoimmune diseases. Here, GWEA is applied to AIHT. The results show that existing epidemiological and genomic data of AIHT adhere to the principles of GWEA. Therefore, AIHT appears to be the result of somatic mutations in people at risk for the disease. AIHT develops once sufficient mutations create a new "autoimmune pathway" driven by non-self-signal and supported by neopeptide formation and signal amplification. Given the random nature of somatic mutations throughout life, the new theory explains why some people with AIHT develop additional autoimmune diseases, why family members may develop a range of non-AIHT autoimmune diseases, why the age of onset cannot be predicted, and why AIHT is transferred to the following generations through dominant inheritance with delayed, incomplete penetrance.

Humans↗

PubMed1998

A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript.

Immunoglobulin (Ig) genes expressed in mature B lymphocytes can undergo somatic hypermutation upon cell interaction with antigen and T cells. The mutation mechanism had previously been shown to depend upon transcription initiation, suggesting that a mutator factor was loaded on an RNA polymerase initiating at the promoter and causing mutations during elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57-65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (EPS) located within the variable (V) region of an Ig transgene. This substrate can easily be assayed for the presence of mutations in DNA from transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites have been altered. Surprisingly, the EPS insert was mutated many times more frequently than the flanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutation where the fine specificity of the mutations is determined by nucleotide sequence preferences of a mutator factor, and where the general site of mutagenesis is determined by the pausing of the RNA polymerase due to secondary structures within the nascent RNA.

Animals↗

PubMed1997

Pattern of usage and somatic hypermutation in the V(H)5 gene segments of a patient with asthma: implications for IgE.

The V(H)5 family contains two functional genes, V5-51 and V(H)32, and appears to be over-represented in IgE antibodies from patients with allergic disease. Previous sequence analysis of V(H)5 gene segments in IgE has revealed a substantial level of somatic hypermutation, with evidence for hotspots. To assess characteristics of V(H)5 gene behavior, V(H)5 gene segments in combination with C mu, C gamma, C alpha, and C epsilon have been amplified from blood B lymphocytes of a patient with atopic asthma. Sequence analysis revealed strong preferential usage of one of the two V(H)5 gene segments (V5-51) by IgM, IgG, and IgA. In contrast, IgE used both genes equally. Levels of somatic mutation were higher following all isotype switches, particularly to IgA. Mutational hotspots were identifiable in all isotypes, leading to several common replacement amino acids. The dominant mutational site in IgM was a common hotspot at Ser31. IgG, IgA, and IgE-derived sequences had mainly common hotspots, with few distinct sites. The results indicate that mutational hotspots are a feature of the V(H)5 gene, are identifiable at an early stage of somatic hypermutation, and are not a unique feature of IgE. Generation of IgE antibodies appears to involve three processes: the preferential use of V(H)5 genes, consistent with superantigen stimulation; the accumulation of somatic mutations in common hotspots, some of which are in complementarity-determining regions (CDR); and the acquisition of non-hotspot mutations in CDR, accounting for approximately 50% of replacement amino acids in these sites, and presumably contributing to affinity maturation.

Adult↗

PubMed2001

Somatic mutation hotspots correlate with DNA polymerase eta error spectrum.

Mutational spectra analysis of 15 immunoglobulin genes suggested that consensus motifs RGYW and WA were universal descriptors of somatic hypermutation. Highly mutable sites, "hotspots", that matched WA were preferentially found in one DNA strand and RGYW hotspots were found in both strands. Analysis of base-substitution hotspots in DNA polymerase error spectra showed that 33 of 36 hotspots in the human polymerase eta spectrum conformed to the WA consensus. This and four other characteristics of polymerase eta substitution specificity suggest that errors introduced by this enzyme during synthesis of the nontranscribed DNA strand in variable regions may contribute to strand-specific somatic hypermutagenesis of immunoglobulin genes at A-T base pairs.

Amino Acid Motifs↗

PubMed1993

Analysis of somatic hypermutation in mouse Peyer's patches using immunoglobulin kappa light-chain transgenes.

We have exploited mice transgenic for an immunoglobulin kappa light chain in order to show that immunoglobulin genes in the B cells of Peyer's patches in unimmunized mice carry a high level of somatic mutations. Most of the mutations are found in the subpopulation of B cells which, based on peanut agglutinin binding, derive from the germinal centers. The number of mutations per clone and their distribution along the variable gene segment (indicative of untemplated point mutations) are very similar to those found in antigen-specific splenic B cells of normal mice after secondary immunization. The mutations accumulate mainly in complementarity-determining region 1, in particular in some specific codons (Ser-26, Ser-31, and Ser-77) which have been previously recognized as intrinsic hypermutational hotspots. These results suggest that, as in the spleen, somatic mutation occurs in B cells which have migrated to the germinal centers, probably as a consequence of stimulation by antigens present in the gut environment. Transgenic animals are increasingly being used to define the signals involved in hypermutation. However, their subsequent study is very time-consuming because it is based on immunization and analysis of hybridomas or antigen-selected cells. We propose that the use of Peyer's patches of unimmunized adult mice offers a reliable and simple approach to analyze hypermutation of transgenes.

Amino Acid Sequence↗

PubMed1998

Diversification of rabbit VH genes by gene-conversion-like and hypermutation mechanisms.

Where, when and how does VH diversification occur in the rabbit? Early diversification by gene-conversion and somatic hypermutation in rabbit appendix and chicken bursa of Fabricius are similar processes; the chicken bursa and the rabbit appendix have homologous functions. However, diversification in bursa starts during embryonic development whereas it starts in rabbit appendix about 2 weeks after birth in the presence of antigens and superantigens that may contribute to positive and negative selection, affect B-cell expansion and mold the repertoire. The biochemical steps leading to diversification by gene conversion are unknown. However elevated levels of RAD51 mRNA in both chicken bursa and young rabbit appendix suggest that repair of double strand breaks may be involved. The base changes found in expressed rabbit VH sequences derived from rearrangement of known germline VH genes followed by one or more gene conversions occur with frequencies similar to those found in analyses of somatic hypermutation. The Ser codons in CDR1 and CDR2 of rabbit VH1 genes are all AGY rather than TCN, suggesting that they may represent intrinsic hotspots for hypermutation comparable to those described in human and mouse VH. Somatic hypermutation may further refine antibody affinities in rabbit germinal centers.

Animals↗

PubMed2000

Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes.

Targeted hypermutation of immunoglobulin variable region genes occurs in B cells during an immune response, and gives rise to families of related mutant antibodies which are then selected for their binding affinity to the immunizing antigen. Somatic hypermutation predominantly generates point mutations, many of which occur at specific residues (hotspots). The reaction has been linked to transcription and requires the presence of immunoglobulin enhancers, but replacement of the variable gene by heterologous sequences, or the variable region promoter by a heterologous promoter, does not interfere with the mutation process. Here we show the existence of abundant DNA double-strand breaks (DSBs) in hypermutating sequences. Generation of the DSBs is coupled to transcription, enhancer-dependent, and correlates with the appearance of nearby mutations. Furthermore, the DSBs are cell-cycle restricted, being found almost exclusively in cells that have completed, or nearly completed, DNA replication. We propose a model for somatic hypermutation in which mutations are introduced into the DNA during repair of DSBs by homologous recombination. The finding of DSBs during somatic hypermutation may help to explain the chromosomal translocations found in some B-cell tumours.

Animals↗

PubMed1996

The targeting of somatic hypermutation.

Somatic hypermutation does not occur randomly within immunoglobulin V genes but, rather, is preferentially targeted to certain nucleotide positions (hot spots) and away from others (cold spots). Cold spots often coincide with residues essential for V gene folding. Hotspots, which appear to be strategically located to favour affinity maturation, are most frequently located in the CDRs (particularly CDR1) though conserved hotspots are also found at the base of FR3. Hotspots are in part created by local DNA sequence and the strong biases of codon usage in V genes indicate that the genes have evolved such that somatic hypermutation is targeted to those parts of the V where it is likely to prove most useful. These features of mutational hotspots and biased codon usage are also evident in V genes of lower animals suggesting that diversification by strategic targeting of non-templated mutation may have evolved early in antigen receptor evolution.

Animals↗

PubMed2006

Somatic hypermutation of Ig genes is affected differently by failures in apoptosis caused by disruption of Fas (lpr mutation) or by overexpression of Bcl-2.

The effects of the two main apoptotic pathways on the somatic hypermutation process were analysed. Transgenic mice carrying the V(kappa)Ox1-J(kappa)5 rat transgene were crossed with Fas-deficient lpr mice or with mice overexpressing the Bcl-2 protein. The transgenic V(kappa)Ox1 segment and the endogenous JH4-C(micro) Ig intron from Peyer's patches germinal centre B cells were sequenced to study the intrinsic somatic hypermutation process without the skewing effects of specific antigen selection. The lpr/ox mice displayed, in both regions, a high level of mutations with a normal pattern of substitutions. On the contrary, the bcl-2/ox mice displayed a lower level of mutations with an altered pattern, showing a decreased mutational rate in the intrinsic hotspots of the V(kappa)Ox1 gene. Our results suggest that the lpr mutation does not have a direct effect on the somatic hypermutation process, but rather on the negative selection of B cells in the germinal centres, leading to the accumulation of recurrent mutations. In contrast, Bcl-2 overexpression might influence the somatic hypermutational process either by altering the incorporation of mutations or by enhancing the repair mechanism(s). The present work supports the hypothesis that both apoptotic pathways, Fas and Bcl-2, play distinct roles in the germinal centre reactions.

Animals↗

PubMed2001

Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation.

After gene rearrangement, immunoglobulin V genes are further diversified by either somatic hypermutation or gene conversion. Hypermutation (in man and mouse) occurs by the fixation of individual, non-templated nucleotide substitutions. Gene conversion (in chicken) is templated by a set of upstream V pseudogenes. Here we show that if the RAD51 paralogues XRCC2, XRCC3 or RAD51B are ablated the pattern of diversification of the immunoglobulin V gene in the chicken DT40 B-cell lymphoma line exhibits a marked shift from one of gene conversion to one of somatic hypermutation. Non-templated, single-nucleotide substitutions are incorporated at high frequency specifically into the V domain, largely at G/C and with a marked hotspot preference. These mutant DT40 cell lines provide a tractable model for the genetic dissection of immunoglobulin hypermutation and the results support the idea that gene conversion and somatic hypermutation constitute distinct pathways for processing a common lesion in the immunoglobulin V gene. The marked induction of somatic hypermutation that is achieved by ablating the RAD51 paralogues is probably a consequence of modifying the recombination-mediated repair of such initiating lesions.

Animals↗

PubMed2006

Trends in antibody sequence changes during the somatic hypermutation process.

Probable germline gene sequences from thousands of aligned mature Ab sequences are inferred using simple computational matching to known V(D)J genes. Comparison of the germline to mature sequences in a structural region-dependent fashion allows insights into the methods that nature uses to mature Abs during the somatic hypermutation process. Four factors determine the residue type mutation patterns: biases in the germline, accessibility from single base permutations, location of mutation hotspots, and functional pressures during selection. Germline repertoires at positions that commonly contact the Ag are biased with tyrosine, serine, and tryptophan. These residue types have a high tendency to be present in mutation hotspot motifs, and their abundance is decreased during maturation by a net conversion to other types. The heavy use of tyrosines on mature Ab interfaces is thus a reflection of the germline composition rather than being due to selection during maturation. Potentially stabilizing changes such as increased proline usage and a small number of double cysteine mutations capable of forming disulfide bonds are ascribed to somatic hypermutation. Histidine is the only residue type for which usage increases in each of the interface, core, and surface regions. The net overall effect is a conversion from residue types that could provide nonspecific initial binding into a diversity of types that improve affinity and stability. Average mutation probabilities are approximately 4% for core residues, approximately 5% for surface residues, and approximately 12% for residues in common Ag-contacting positions, excepting the those coded by the D gene.

Amino Acid Sequence↗