Characterization of Ig gene somatic hypermutation in the absence of activation-induced cytidine deaminase

Somatic hypermutation (SHM) of Ig genes depends upon the deamination of C nucleotides in WRCY (W = A/T, R = A/G, Y = C/T) motifs by activation-induced cytidine deaminase (AICDA). Despite this, a large number of mutations occur in WA motifs that can be accounted for by the activity of polymerase eta (POL eta). To determine whether there are AICDA-independent mutations and to characterize the relationship between AICDA- and POL eta-mediated mutations, 1470 H chain and 1313 kappa- and lambda-chain rearrangements from three AICDA(-/-) patients were analyzed. The Ig mutation frequency of all V(H) genes from AICDA(-/-) patients was 40-fold less than that of normal donors, whereas the mutation frequency of mutated V(H) sequences from AICDA(-/-) patients was 6.8-fold less than that of normal donors. AICDA(-/-) B cells lack mutations in WRCY/RGYW motifs as well as replacement mutations and mutational targeting in complementarity-determining regions. A significantly reduced mutation frequency in WA motifs compared with normal donors and an increased percentage of transitions, which may relate to reduced uracil DNA-glycosylase activity, suggest a role for AICDA in regulating POL eta and uracil DNA-glycosylase activity. Similar results were observed in V(L) rearrangements. The residual mutations were predominantly G:C substitutions, indicating that AICDA-independent cytidine deamination was a likely, yet inefficient, mechanism for mutating Ig genes.     

The targeting of somatic hypermutation closely resembles that of meiotic mutation

We have compared the microsequence specificity of mutations introduced during somatic hypermutation (SH) and those introduced meiotically during neutral evolution. We have minimized the effects of selection by studying nonproductive (hence unselected) Ig V region genes for somatic mutations and processed pseudogenes for meiotic mutations. We find that the two sets of patterns are very similar: the mutabilities of nucleotide triplets are positively correlated between the somatic and meiotic sets. The major differences that do exist fall into three distinct categories: 1) The mutability is sharply higher at CG dinucleotides under meiotic but not somatic mutation. 2) The complementary triplets AGC and GCT are much more mutable under somatic than under meiotic mutation. 3) Triplets of the form WAN (W = T or A) are uniformly more mutable under somatic than under meiotic mutation. Nevertheless, the relative mutabilities both within this set and within the SAN (S = G or C) triplets are highly correlated with those under meiotic mutation. We also find that the somatic triplet specificity is strongly symmetric under strand exchange for A/T triplets as well as for G/C triplets in spite of the strong predominance of A over T mutations. Thus, we suggest that somatic mutation has at least two distinct components: one that specifically targets AGC/GCT triplets and another that acts as true catalysis of meiotic mutation.     

Guanine Holes Are Prominent Targets for Mutation in Cancer and Inherited Disease

Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G•C bp in the context of all 64 5′-NGNN-3′ motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials). Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.     

SAIVGeM: spreadsheet analysis of immunoglobulin VH gene mutations

The analysis of mutations in immunoglobulin heavy chain variable (IGHV) region genes is a tedious process when performed by hand on multiple sequences. This report describes a set of linked Microsoft Excel files that perform several common analyses on large numbers of IGHV sequences. The spreadsheet analysis of immunoglobulin VH gene mutations (SAIVGeM) package determines the distribution of mutations among each nucleotide, the nature of the mutation at both the nucleotide and amino acid level, the frequency of mutation in the A/G G C/T A/T (RGYW) hotspot motifs of both strand polarity, and the distribution of replacement and silent mutations among the complementarity determining regions (CDRs) and the framework regions (FRs) of the immunoglobulin gene as defined by either the Kabat or IMGT conventions. These parameters are summarized and graphically presented where appropriate. In addition, the SAIVGeM package analyzes those mutations that occur in third positions of redundant codons. Because any nucleotide change in these positions is inherently silent, these positions can be used to study the mutational spectra without biases from the selection of protein structure.     

HotSpotter: efficient visualization of driver mutations

Background

Driver mutations are positively selected during the evolution of cancers. The relative frequency of a particular mutation within a gene is typically used as a criterion for identifying a driver mutation. However, driver mutations may occur with relative infrequency at a particular site, but cluster within a region of the gene. When analyzing across different cancers, particular mutation sites or mutations within a particular region of the gene may be of relatively low frequency in some cancers, but still provide selective growth advantage.

Results

This paper presents a method that allows rapid and easy visualization of mutation data sets and identification of potential gene mutation hotspot sites and/or regions. As an example, we identified hotspot regions in the NFE2L2 gene that are potentially functionally relevant in endometrial cancer, but would be missed using other analyses.

Conclusions

HotSpotter is a quick, easy-to-use visualization tool that delivers gene identities with associated mutation locations and frequencies overlaid upon a large cancer mutation reference set. This allows the user to identify potential driver mutations that are less frequent in a cancer or are localized in a hotspot region of relatively infrequent mutations.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1044) contains supplementary material, which is available to authorized users.     

Mutation pattern of immunoglobulin transgenes is compatible with a model of somatic hypermutation in which targeting of the mutator is linked to the direction of DNA replication.

We have previously demonstrated that B lymphocyte specific somatic mutations are introduced into the variable regions of immunoglobulin kappa transgenes in two independent transgenic mouse lines. The frequency, distribution and nature of these mutations strongly suggest that they arose as a result of the process of somatic hypermutation, which is responsible, in part, for affinity maturation during an immune response. Unexpectedly, in these multiple copy transgenic lines, many of the transgene copies showed no evidence of somatic mutation. This paradox was addressed by determining the sequence of each transgene copy in several B cell hybridomas derived from a mouse line carrying three copies of the kappa transgene. It was found that the somatic hypermutation process in different B cells from the same mouse preferentially targets one, but not the same, transgene copy. We present a model, based on the pattern of this targeting, which links somatic hypermutation to the orientation of the Ig gene relative to the direction of DNA replication.     

Hypermutation at A-T base pairs: the A nucleotide replacement spectrum is affected by adjacent nucleotides and there is no reverse complementarity of sequences flanking mutated A and T nucleotides

Hypermutation is thought to be a two-phase process. The first phase is via the action of activation-induced cytidine deaminase (AID), which deaminates C nucleotides in WRC motifs. This results in the RGYW/WRCY hot spot motifs for mutation from G and C observed in vivo. The resemblance between the hot spot for C mutations and the reverse complement of that for G mutations implies a process acting equally on both strands of DNA. The second phase of hypermutation generates mutations from A and T and exhibits strand bias, with more mutations from A than T. Although this does not concur with the idea of one mechanism acting equally on both strands, it has been suggested that the AT mutator also has a reversible motif; WA/TW. We show here that the motifs surrounding the different substitutions from A vary significantly; there is no single targeting motif for all A mutations. Sequence preferences associated with mutations from A more likely reflect an influence of adjacent nucleotides over what the A mutates "to." This influence tends toward "like" replacements: Purines (A or G) in the 5' position bias toward replacement by another purine (G), whereas replacement with pyrimidines (C or T) is more likely if the preceding base is also a pyrimidine. There is no reverse complementarity in these observations, in that similar influences of nucleotides adjacent to T are not seen. Hence, WA and TW should not be considered as reverse complement hot spot motifs for A and T mutations.     

DNA polymerase η mutational signatures are found in a variety of different types of cancer

DNA polymerase (pol) η is a specialized error-prone polymerase with at least two quite different and contrasting cellular roles: to mitigate the genetic consequences of solar UV irradiation, and promote somatic hypermutation in the variable regions of immunoglobulin genes. Misregulation and mistargeting of pol η can compromise genome integrity. We explored whether the mutational signature of pol η could be found in datasets of human somatic mutations derived from normal and cancer cells. A substantial excess of single and tandem somatic mutations within known pol η mutable motifs was noted in skin cancer as well as in many other types of human cancer, suggesting that somatic mutations in A:T bases generated by DNA polymerase η are a common feature of tumorigenesis. Another peculiarity of pol ηmutational signatures, mutations in YCG motifs, led us to speculate that error-prone DNA synthesis opposite methylated CpG dinucleotides by misregulated pol η in tumors might constitute an additional mechanism of cytosine demethylation in this hypermutable dinucleotide.     

Partial Restoration of Replication of Simian Immunodeficiency Virus by Point Mutations in either the Dimerization Initiation Site (DIS) or Gag Region after Deletion Mutagenesis within the DIS

We used the simian immunodeficiency virus (SIV) molecular clone SIVmac239 to generate a deletion construct, termed SD2, in which we eliminated 22 nucleotides at positions +398 to +418 within the putative dimerization initiation site (DIS) stem. This SD2 deletion severely impaired viral replication, due to adverse effects on the packaging of viral genomic RNA, the processing of Gag proteins, and viral protein patterns. However, long-term culture of SD2 in either C8166 or CEMx174 cells resulted in restoration of replication capacity, due to two different sets of three compensatory point mutations, located within both the DIS and Gag regions. In the case of C8166 cells, both a K197R and a E49K mutation were identified within the capsid (CA) protein and the p6 protein of Gag, respectively, while the other point mutation (A423G) was found within the putative DIS loop. In the case of CEMx174 cells, two compensatory mutations were present within the viral nucleocapsid (NC) protein, E18G and Q31K, in addition to the same A423G substitution as observed with C8166 cells. A set of all three mutations was required in each case for restoration of replication capacity, and either set of mutations could be substituted for the other in both the C8166 and CEMx174 cell lines.     

Chromatin accessibility shapes meiotic recombination in mouse primordial germ cells through assisting double-strand breaks and loop formation

Meiotic recombination is a driver of evolution, and aberrant recombination is a major contributor to aneuploidy in mammals. Mechanism of recombination remains elusive yet. Here, we present a computational analysis to explore recombination-related dynamics of chromatin accessibility in mouse primordial germ cells (PGCs). Our data reveals that: (1) recombination hotspots which get accessible at meiosis-specific DNase I-hypersensitive sites (DHSs) only when PGCs enter meiosis are located preferentially in intronic and distal intergenic regions; (2) stable DHSs maintained stably across PGC differentiation are enriched by CTCF motifs and CTCF binding and mediate chromatin loop formation; (3) compared with the specific DHSs aroused at meiotic stage, stable DHSs are largely encoded in DNA sequence and also enriched by epigenetic marks; (4) PRDM9 is likely to target nucleosome-occupied hotspot regions and remodels local chromatin structure to make them accessible for recombination machinery; and (5) cells undergoing meiotic recombination are deficient in TAD structure and chromatin loop arrays are organized regularly along the axis formed between homologous chromosomes. Taken together, by analyzing DHS-related DNA features, epigenetic marks and 3D genome structure, we revealed some specific roles of chromatin accessibility in recombination, which would expand our understanding of recombination mechanism.     

AID,APOBEC3A and APOBEC3B efficiently deaminate deoxycytidines neighboring DNA damage induced by oxidation or alkylation

BackgroundAID/APOBEC3 (A3) enzymes instigate genomic mutations that are involved in immunity and cancer. Although they can deaminate any deoxycytidine (dC) to deoxyuridine (dU), each family member has a signature preference determined by nucleotides surrounding the target dC. This WRC (W = A/T, R = A/G) and YC (Y = T/C) hotspot preference is established for AID and A3A/A3B, respectively. Base alkylation and oxidation are two of the most common types of DNA damage induced environmentally or by chemotherapy. Here we examined the activity of AID, A3A and A3B on dCs neighboring such damaged bases.MethodsSubstrates were designed to contain target dCs either in normal WRC/YC hotspots, or in oxidized/alkylated DNA motifs. AID, A3A and A3B were purified and deamination kinetics of each were compared between substrates containing damaged vs. normal motifs.ResultsAll three enzymes efficiently deaminated dC when common damaged bases were present in the -2 or -1 positions. Strikingly, some damaged motifs supported comparable or higher catalytic efficiencies by AID, A3A and A3B than the WRC/YC motifs which are their most favored normal sequences. Based on the resolved interactions of AID, A3A and A3B with DNA, we modeled interactions with alkylated or oxidized bases. Corroborating the enzyme assay data, the surface regions that recognize normal bases are predicted to also interact robustly with oxidized and alkylated bases.ConclusionsAID, A3A and A3B can efficiently recognize and deaminate dC whose neighbouring nucleotides are damaged.General significanceBeyond AID/A3s initiating DNA damage, some forms of pre-existing damaged DNA can constitute favored targets of AID/A3s if encountered.     

Review of the molecular characteristics of gene mutations of the germline and somatic cells of the human

Molecular analyses of the limited number of de novo germinal mutations identified in humans indicate that an array of alterations in gene structure can be generated. Similar conclusions are derived from the large data set obtained from molecular analyses of alleles that segregate in the human population and cause genetic diseases. The molecular alterations include nucleotide substitutions as well as insertions, deletions and other rearrangements of the DNA. The lesions may be located in the coding or the noncoding regions of genes or may involve the flanking sequences. The insertions and deletions involve fragments ranging from single nucleotides to many kilobases, and involve both unique sequences and repetitive elements. The nature of the lesions observed to date as either de novo mutations or segregating variants suggests there are locus-specific characteristics of the alterations in DNA structure that are recovered as genetic diseases. Differences in mutation spectra among genetic loci appear to reflect both the structure of the target sequences and the relationship between gene structure and gene function. No induced germinal mutations have been identified, thus no data are available that reveal the relationships between mutagenic exposures and the molecular fingerprints of the lesion induced in the human germ cell and transmitted to the subsequent generations. In contrast, the prospects for analyzing the roles of genetic target, exposure history and individual responsiveness to exposure in creating particular molecular lesions in somatic cells are excellent, both for alterations of single nucleotides and for major alterations of gene structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Integrative Analysis of Somatic Mutations Altering MicroRNA Targeting in Cancer Genomes

Determining the functional impact of somatic mutations is crucial to understanding tumorigenesis and metastasis. Recent sequences of several cancers have provided comprehensive lists of somatic mutations across entire genomes, enabling investigation of the functional impact of somatic mutations in non-coding regions. Here, we study somatic mutations in 3′UTRs of genes that have been identified in four cancers and computationally predict how they may alter miRNA targeting, potentially resulting in dysregulation of the expression of the genes harboring these mutations. We find that somatic mutations create or disrupt putative miRNA target sites in the 3′UTRs of many genes, including several genes, such as MITF, EPHA3, TAL1, SCG3, and GSDMA, which have been previously associated with cancer. We also integrate the somatic mutations with germline mutations and results of association studies. Specifically, we identify putative miRNA target sites in the 3′UTRs of BMPR1B, KLK3, and SPRY4 that are disrupted by both somatic and germline mutations and, also, are in linkage disequilibrium blocks with high scoring markers from cancer association studies. The somatic mutation in BMPR1B is located in a target site of miR-125b; germline mutations in this target site have previously been both shown to disrupt regulation of BMPR1B by miR-125b and linked with cancer.     

An in vivo approach to identifying sequence context of 8-oxoguanine mutagenesis

Base substitution mutations are not distributed randomly in that most are located at a few specific hotspots sites. We have been studying 7,8-dihydro-8-oxoguanine mutagenesis in Escherichia coli in the supF gene carried in a plasmid. Among hotspots, guanine within the 5'-AGA-3' located in the anticodon site was susceptible to the induction of G:C-->T:A transversion. In this study, we constructed variants of the supF gene in which the hotspot 5'-AGA-3' was modified to 5'-AGT-3', 5'-AGG-3' and 5'-AGC-3' to determine the influence of 3' neighboring base on G:C-->T:A mutational activity. Using these variant supF genes propagated in a 7,8-dihydro-8-oxoguanine repair-deficient host, we found that guanine within 5'-AGA-3' and 5'-AGG-3' produce G:C-->T:A, but guanine within 5'-AGT-3' and 5'-AGC-3' reduce the formation of G:C-->T:A. These changes were thus due to the effect of sequence context on the efficiency of mutation formation at the sites of 7,8-dihydro-8-oxoguanine. We also observed a longer range base-pair effect on hotspot formation.     

The Cancer Mutation D83V Induces an α-Helix to β-Strand Conformation Switch in MEF2B

MEF2B is a major target of somatic mutations in non-Hodgkin lymphoma. Most of these mutations are non-synonymous substitutions of surface residues in the MADS-box/MEF2 domain. Among them, D83V is the most frequent mutation found in tumor cells. The link between this hotspot mutation and cancer is not well understood. Here we show that the D83V mutation induces a dramatic α-helix to β-strand switch in the MEF2 domain. Located in an α-helix region rich in β-branched residues, the D83V mutation not only removes the extensive helix stabilization interactions but also introduces an additional β-branched residue that further shifts the conformation equilibrium from α-helix to β-strand. Cross-database analyses of cancer mutations and chameleon sequences revealed a number of well-known cancer targets harboring β-strand favoring mutations in chameleon α-helices, suggesting a commonality of such conformational switch in certain cancers and a new factor to consider when stratifying the rapidly expanding cancer mutation data.