Hypermutation Process Research Articles

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.

Somatic mutations and activation-induced cytidine deaminase (AID) expression in established rheumatoid factor-producing lymphoblastoid cell line

Epstein-Barr virus (EBV) transforms human peripheral B cells into lymphoblastoid cell lines (LCLs), allowing the production of specific antibody-secreting cell lines. We and others have previously found that in contrast to peripheral blood B cells, EBV-transformed lymphoblastoid cell lines express the activation-induced cytidine deaminase (AID) gene. The opposite is true for the germinal center-specific BCL6 gene: it is expressed in adult peripheral blood B cells and is no longer expressed in LCLs. The present work extends our findings and shows that whereas AID expression is rapidly induced following EBV infection, BCL6 expression is gradually down-regulated and is fully extinguished in already established LCLs. The question of whether AID activation induces the process of somatic hypermutation (SHM) was investigated in adult-derived LCLs. It was found that the V H gene from the rheumatoid factor-producing RF LCL (derived from a rheumatoid arthritis patient), accumulated somatic point mutations in culture. Overall, nine unique mutations have accumulated in the rearranged V H gene since the generation of the RF cell line. Four additional intraclonal mutations were found among 10 cellular clones of the RF cells. One out of the four was in CDR1 and could be correlated with loss of antigen-binding activity in three out of the 10 clones. Altogether, these 13 mutations were preferentially targeted to the DGYW motifs and showed preference for CG nucleotides, indicating that they were AID-mediated. By contrast, mutations were not detected among 3700–4000 nucleotides each of the Vλ, Cμ and GAPDH genes derived from the same RF cell cultures and the cellular clones. Our results thus show that AID may generate point mutations in the rearranged Ig V H during in vitro cell culture of adult-LCLs and that these mutations may be responsible, at least in part, for the known instability and occasional loss of antigen-binding activity of antibody-secreting LCLs.

Antibody Quality in Old Age

There is very little change in the quantity of antibodies people produce, of any isotype, with age. However, there is a change in the quality of the antibody response. Older people produce fewer antibodies that are specific for the activating pathogen or vaccine. At the same time, the number of nonspecific antibodies increases. Quite often these antibodies have self-reactivity (e.g., anti-dsDNA). The appearance of these antibodies is not associated with pathogenic autoimmune disease, although it is true that the incidence of some autoimmune diseases increases with age. The authors postulate that the process of antibody affinity maturation is compromised in old age. No evidence was found that the process of hypermutation is compromised with age. However, using graph theory to study the dynamics of a germinal center selection process, a decrease in the extent of selection occurring in the germinal centers of mucosal tissue was observed with age. This is a tissue-specific phenomenon because the decrease was not seen in the germinal centers of spleen. Because selection of highly specific cells in the germinal center depends on a number of factors (number and quality of founder cells, help from T cells, and follicular dendritic cells) these need to be investigated further to determine what is needed to improve the affinity mutation process.

Rescue of “crippled” germinal center B cells from apoptosis by Epstein-Barr virus

Epstein-Barr virus (EBV) is associated with B-cell lymphomas such as Hodgkin lymphoma, Burkitt lymphoma, and post-transplantation lymphoma, which originate from clonal germinal center (GC) B cells. During the process of somatic hypermutation, GC B cells can acquire deleterious or nonsense mutations in the heavy and light immunoglobulin genes. Such mutations abrogate the cell surface expression of the B-cell receptor (BCR), which results in the elimination of these nonfunctional B cells by immediate apoptosis. EBV encodes several latent genes, among them latent membrane protein 1 (LMP1) and LMP2A, which are regularly expressed in EBV-positive Hodgkin lymphoma and posttransplantation lymphomas. Since LMP1 and LMP2A mimic the function of 2 key receptors on B cells, CD40 and BCR, respectively, we wanted to learn whether EBV infection can rescue proapoptotic GC B cells with crippling mutations in the heavy chain immunoglobulin locus from apoptosis. We show here that BCR-negative GC B cells readily enter the cell cycle upon infection with EBV in vitro and yield clonal lymphoblastoid cell lines that are incapable of expressing a functional BCR because the rearranged and formerly functional heavy chain immunoglobulin alleles carry deleterious mutations. Our findings imply an important role for EBV in the process of lymphomagenesis in certain cases of Hodgkin lymphoma and posttransplantation lymphomas.

Transformation of BCR-deficient germinal-center B cells by EBV supports a major role of the virus in the pathogenesis of Hodgkin and posttransplantation lymphomas

In classic Hodgkin lymphoma (HL) and posttransplantation lymphoproliferative disease (PTLD), 2 malignancies frequently associated with Epstein-Barr virus (EBV), the tumor cells often appear to derive from B-cell receptor (BCR)-deficient and therefore preapoptotic germinal center (GC) B cells. To test whether EBV can rescue BCR-less GC B cells, we infected human tonsillar CD77+ GC B cells in vitro with EBV. More than 60 monoclonal lymphoblastoid cell lines (LCLs) were established. Among these, 28 cell lines did not express surface immunoglobulin (sIg). Two of the sIg-negative cell lines carry obviously destructive mutations that have been introduced into originally functional V(H) gene rearrangements during the process of somatic hypermutation. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) showed that in most other lines the sIg deficiency was not simply the result of transcriptional down-regulation, but it was rather due to posttranscriptional defects. These findings strongly support the idea that EBV plays a central role in the pathogenesis of classic HL and PTLD by rescuing BCR-deficient, preapoptotic GC B cells from apoptosis, and that EBV infection renders the cells independent from survival signals normally supplied by a BCR. The monoclonal LCLs represent valuable models for early stages of lymphoma development in classic HL and PTLD.

Epstein-Barr virus infection in vitro can rescue germinal center B cells with inactivated immunoglobulin genes

Immunoglobulin genotyping of Epstein-Barr virus (EBV)-positive posttransplantation lymphoproliferative disease has suggested that such lesions often arise from atypical post-germinal center B cells, in some cases carrying functionally inactivated immunoglobulin genes. To investigate whether EBV can rescue cells that are failed products of the somatic hypermutation process occurring in germinal centers (GCs), we isolated GC cells from tonsillar cell suspensions and exposed them to EBV in vitro. Screening more than 100 EBV-transformed cell lines of GC origin identified 6 lines lacking surface immunoglobulin, a phenotype never seen among lines derived from circulating naive or memory B cells. Furthermore, 3 of the 6 surface immunoglobulin-negative GC lines carried inactivating mutations in the immunoglobulin H (IgH) variable gene sequence. The ability of EBV to rescue aberrant products of the germinal center reaction in vitro strengthens the probability that a parallel activity contributes to EBV's lymphomagenic potential in vivo.

Expression of Specialized Error-Prone DNA Polymerases in Chronic Lymphocytic Leukemia (CLL).

Specialized DNA polymerases are low fidelity enzymes required to bypass DNA lesions that are also involved in somatic hypermutation. Deregulation of these enzymes can lead both to an increased mutation rate and to an enhanced translesion synthesis capability. DNA polymerase Kappa (DNA pol Kappa) has been found to promote tumorigenesis and to be overexpressed in some cancers. Expression levels of specialized DNA polymerases Iota, Eta, Zeta and Mu were analyzed in primary B-cells from 12 CLL patients (6 with IgVH mutated genes [M] and 6 with unmutated [UM] genes). Expression of DNA pol Kappa was also assessed in 27 CLL samples (13 M and 14 UM), in B-cell lines derived from pre- and post-germinal center malignancies (NALM-6 [preB-acute lymphoblastic leukemia], JVM-2 [prolymphocytic leukemia], Granta, NCEB and REC [mantle-cell lymphoma] and Daudi [Burkitt's lymphoma] and in normal B-cells from peripheral blood and tonsil. Expression of DNA polymerases was measured by Quantitative RT-PCR using Ramos (a constitutively hypermutating Burkitt's lymphoma cell line) as a reference, considering an arbitrary unit of 1 for each gene. Although somatic hypermutation is absent in UM CLL cells, there were no differences in the expression of the DNA polymerases analyzed according to the mutational status. Expression of DNA pols Iota and Zeta was similar in CLL cells and Ramos (mean value: 0.95 and 1.3, respectively), whereas expression of DNA pol Mu was slightly higher (mean 3.24) and DNA pol Eta was underexpressed (mean 0.19). In contrast, among all the hematological malignancies analyzed, significant overexpression of DNA pol Kappa was only found in CLL samples (mean value; 8.43;range: 1.42–19.47). Induction of AID expression by stimulating CLL cells with CD40 ligand and IL-4 did not modulate DNA pol Kappa expression. Finally, expression of DNA pol Kappa in normal B cells from peripheral blood was similar to that in Ramos (0.87) and slightly higher in B cells from tonsil (2.02). In conclusion, no differences in DNA polymerases were observed between M and UM CLL. As previously described in some solid tumors, CLL cells showed an increased expression of DNA pol Kappa, regardless the mutational status of the IgVH genes, suggesting that the expression of this polymerase is not related to the process of somatic hypermutation that occurs in some CLL cells. Taken together, these results support the concept that DNA pol Kappa may behave as an oncogene in CLL and that its regulation may be crucial for maintaining genomic stability.

A Very Short Mutational Hot-Spot in the IGH 5′Sμ Region Suggests That the Acquisition of a Class Switch Recombination Defect Is an Important Step Toward Lymphomagenesis.

The t(14;18), which occurs during VDJ joining, is usually seen as the primary genetic event in follicular lymphoma (FL). Pre-lymphoma cells would then migrate to the secondary lymphoid organs and acquire secondary genetic abnormalities. The mechanisms leading to these secondary lesions are unclear, but both somatic hypermutation (SHM) and class switch recombination (CSR) processes may be involved. Surprisingly, while many FL still express an IgM, aberrant switch junctions were reported on both IGH alleles, indicating dysfunctions of CSR during the onset of the neoplasm. Recently, AID mediated point mutations were shown to accumulate in the switch μ (Sμ) regions of stimulated B cells undergoing CSR. To get insights into a possible CSR deregulation in FL, we sequenced the 5′ end of the Sμ region for 73 t(14;18) positive tumours. 152 point mutations and 5 deletions were identified, for a mutation frequency of 0.31/100 bp. Notably, mutations were found for a majority of cases (49/73, 67.1%) rather than being restricted to few tumours. Significant targeting of RGYW motifs (p<10−4) and bias for transitions over transvertions (p<10−4) both suggested the involvement of AID. More importantly, our data showed a highly significant clustering effect, with 22 mutations (13,9%) found on a 4 bp cluster on the GTAA splicing donor site of the Iμ exon (p<10−6). Those 22 mutations were found in different tumors so that 30.1% of the lymphomas in our series had a mutated splicing site. Analysis of whole blood genomic DNA from 14 patients and 20 healthy donors indicated that these mutations are not polymorphisms but are instead acquired by tumor cells. Importantly, allele specific PCR experiments showed no clustering effect on the translocated alleles, while 15/79 (19%) mutations on the functional alleles were found on the Iμ splicing site (p<10−6). Such mutations were also found, but on the translocated alleles, for two additional lymphomas with complex t(3;14;18)(q27;q32;q21) and t(8;14;18)(q24;q32;q21) translocations, suggesting that they may be linked to downstream rearrangements on the IGH locus. Finally, RT-PCR experiments showed that a mutation of the Iμ splicing site leads to the expression of aberrant germline transcripts. Together, our data thus indicate that mutations leading to an incorrect splicing of the germline Sμ transcripts are probably positively selected during lymphomagenesis, and therefore that an acquired perturbation of the CSR process may have a major impact during the onset of the disease, maybe by preventing pre-lymphoma cells from switching their surface immunoglobulin and/or by favouring subsequent genetic lesions.

Nucleotide Insertions and Deletions in Chronic Lymphocytic Leukemia. A CLL Specific Deletion among IGHV3-21 Expressing Cases with Stereotyped Receptors.

Insertions/duplications and deletions (I/D/Ds) in IG variable region genes are infrequent sequelae of the somatic hypermutation process. In the present study, we document the occurrence of nucleotide I/D/Ds in rearranged IGHV genes in CLL patients. Among 809 IGHV-D-J sequences amplified in 760 CLL cases, 9 (1.11%) and 15 (1.85%) sequences exhibited IGHV sequence changes consistent with nucleotide insertions/duplications or deletions, respectively. I/D/Ds were found in genes of the IGHV1, IGHV3 and IGHV4 subgroups, always within mutated IGHV-D-J rearrangements. In 21/24 cases, the inserted/duplicated or lost nucleotides occurred in multiples of 3; therefore, the original reading frame was maintained and a potentially intact receptor was coded. I/D/Ds were located completely or in part within CDRs in 21/24 cases; sequence motifs (AGY, AGA, AAC trinucleotides) that resemble intrinsic hotspots for somatic hypermutation were identified in 21/24 sequences. Short stretches with high homology (misalignment feet) that would offer the DNA polymerase an alternative template for re-annealing in cases of replication slippage were identified in all CLL sequences carrying I/D/Ds. I/D/Ds were generated somatically since (i) they always occurred in cases with somatic mutations, (ii) they could not be found on sequencing analysis of the corresponding germline IGHV genes amplified on DNA isolated from selected patients' neutrophils, thus excluding a possible genetic polymorphism. The incidence of I/D/Ds in CLL is consistent with previous reports in normal, autoreactive and neoplastic human B cells, thus seemingly indicating that these modifications generally arise without any particular disease-specific associations. A striking exception to this rule was identified in the case of CLL IGHV3-21 expressing cases: one aminoacid was deleted from the CDR2 region in 16/74 (21.6%) IGHV3-21 CLL sequences (database-derived IGHV3-21 CLL cases + present series) vs. only 2/340 (0.59%) non-CLL IGHV3-21 sequences; 15/16 CLL IGHV3-21 sequences carrying this deletion belonged to a subset with unique, shared HCDR3s and light chain CDR3 motifs. The effect of the CDR2 deletion on the structure of the IG molecules in this subset of CLL patients was studied with molecular modelling and dynamics simulations. The models suggested that the deletion could be accommodated without significantly affecting the local structure. The close association of deletions in CDR2 with the homologous subset of IGHV3-21 expressing CLL cases provides further evidence for the importance of an antigenic drive in malignant transformation and/or maintenance of the neoplastic clone in at least some CLL cases.

Aberrant Somatic Hypermutation in Extranodal Marginal Zone B-Cell Lymphoma of MALT Type.

Extranodal marginal zone B-cell lymphoma of mucosa associated lymphoid tissue (MALT lymphoma) accounts for approximately 7% to 8% of all non-Hodgkin lymphomas (NHLs) being the third most frequent histological subtype. The gastrointestinal tract - particularly the stomach - is the most common site of MALT lymphoma comprising 50% of all cases, but virtually every organ may be affected by this type of lymphoma. Transformation (or de novo emergence at extranodal sites) in diffuse large B-cell lymphoma (DLBCL) occurs but - according to the WHO criteria - is considered as separate entity. The understanding of the molecular biology of MALT lymphoma has significantly improved following the recent cloning of recurrent balanced translocations such as t(11;18) or t(14;18), but a mechanism for genome-wide instability during MALT lymphomagenesis has not been described. We have reported that the somatic hypermutation process (SHM) physiologically aimed at mutating the immunoglobulin variable gene (IgV) aberrantly targets multiple proto-oncogenes in >50% of DLCBL (Pasqualucci et al., Nature 412:341, 2001). Consequently, multiple mutations are introduced in the 5′ region of genes including known proto-oncogenes such as PIM-1, PAX-5, Rho/TTF and c-MYC. To further investigate whether aberrant somatic hypermutation (ASHM) also occurs in MALT lymphoma, we studied the mutation profile of these genes in 17 MALT lymphomas (6 of gastric- and 11 of nongastric origin) and 18 extranodal DLBCL (10 gastric, 8 nongastric). Mutations in one or more genes were detected in 15 of 17 (88.2%) cases of MALT lymphoma and in all of 18 (100%) cases of extranodal DLBCL. 7 of 17 (41.2%) and 15 of 18 (83.3%) carried mutations in two or more genes in the MALT- and DLBC-lymphoma group, respectively. Overall, mutations in PIM-1 occurred in 5 of 17 (29.4%) cases with MALT lymphoma and in 10 of 18 (55.5%) in extranodal DLBCL cases. For PAX-5, the distribution of mutated cases between MALT- and DLBC-lymphoma was 6 of 17 (35.3%) and 10 of 18 (55.5%), for Rho/TTF 3 of 17 (17.6%) and 8 of 18 (44.4%) and for c-MYC 9 of 17 (52.9%) and 12 of 18 (66.6%), respectively. A total of 99 sequence variants were found in 35 cases, 29 in the MALT lymphomas and 70 in extranodal DLBCL. Although the mutations were almost exclusively single base pair substitutions (n=98 ), an insertion was also present (n=1). Mutations were of somatic origins, occur independent of chromosomal translocations to the Ig loci and share features of the IgV SHM process including bias for transition over transversion, preferential hotspot (RGYW/WRCY) targeting and restriction to the first 1–2Kb from the promoter. The mean mutation frequency in mutated MALT lymphomas was with 0.045 x10−2/bp 1.7 fold lower compared to 0.08 x10−2/bp in mutated extranodal DLBCL. Further in PIM-1, PAX-5 and c-MYC some of the mutations were found to affect coding exons, leading to amino acid exchanges, thus potentially altering gene function. These data indicate that aberrant SHM is associated with extranodal DLBCL and MALT lymphoma, likewise. By mutating regulatory and coding sequences of the targeted genes and by possibly favouring chromosomal translocations ASHM may represent a major contributor to their pathogenesis. ASHM may further support a model of MALT lymphomagenesis leading from an antigen driven lesion to transformed MALT lymphoma finally evolving to overt DLBCL.

Antibody repertoire development in the sheep

The model of immunoglobulin (Ig) repertoire diversification in sheep has evolved dramatically in recent years. A process thought to involve the rearrangement of a very limited number of variable (V), diversity (D) and joining (J) segments followed by intense, antigen (Ag)-independent, somatic hypermutation is now known to be less recombinatorially restrictive and to involve fewer mutational events. Although mutation rates are now lower than previously thought, the somatic hypermutation process itself is no less critical to the development of the primary Ig repertoire in sheep. Recent studies have shown that those B cells that fail to mutate will die via apoptosis. Much of the V(D)J rearrangement is thought to occur in the fetal liver and spleen prior to development of the ileal Peyer's patch (PP) at approximately day 100 of gestation. Although de novo Ig rearrangement likely does not occur in the ileal PP, this tissue is a site of massive B-cell proliferation, selection and Ig diversification through somatic hypermutation.

The evolution of human anti-double-stranded DNA autoantibodies

It has been proposed that the anti-double-stranded DNA (dsDNA) response in patients with systemic lupus erythematosus (SLE) is antigen driven and that DNA or nucleosomes select anti-DNA reactive, somatically mutated B cells. We have used site-directed mutagenesis to systematically revert the somatic mutations of two human anti-dsDNA antibodies from SLE patients to analyze the resulting changes in DNA binding as well as binding to other autoantigens. Our data demonstrate that high-affinity binding to dsDNA and nucleosomes is acquired by somatic replacement mutations in a stepwise manner. Reactivity to surface structures of apoptotic cells is acquired by the same somatic mutations that generate high-affinity dsDNA binding. Importantly, revertant antibodies with germ-line V regions did not show any measurable DNA reactivity. We propose that anti-DNA autoantibodies are generated from nonautoreactive B cells during a normal immune response. B cells may acquire autoreactivity de novo during the process of somatic hypermutation. Nucleosomes, if available in lupus patients because of defects in clearing of apoptotic debris, might subsequently positively select high affinity anti-DNA B cells.

Polymerase μ is up‐regulated during the T cell‐dependent immune response and its deficiency alters developmental dynamics of spleen centroblasts

Mammalian DNA polymerase mu (Polmu), preferentially expressed in secondary lymphoid organs, is shown here to be up-regulated in germinal centers after immunization. Alternative splicing appears to be part of Polmu regulation during an immune response. We generated Polmu-deficient mice that are viable and show no anatomical malformation or serious alteration in lymphoid populations, with the exception of an underrepresentation of the B cell compartment. Young and aged homozygous Polmu(-/-) mice generated similar immune responses after immunization with the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to chicken gammaglobulin (CGG), compared with their wild-type littermates. Nonetheless, the kinetics of development of the centroblast population showed significant differences. Hypermutation analysis of the rearranged heavy chain intron region in centroblasts isolated from NP-CGG-immunized Polmu(-/-) mice showed a similar quantitative and qualitative somatic mutation spectrum, but a lower representation of heavily mutated clones. These results suggest that although it is not a critical partner, Polmu modulates the in vivo somatic hypermutation process.

Hypermutation Rate Normalized by Chronological Time

It is generally believed that in cells undergoing Ig somatic hypermutation, more cell divisions result in more mutations. This is because DNA synthesis and replication is thought to play roles in the known mechanisms-cytidine deamination and subsequent conversion to thymidine, uracil-DNA glycosylase-mediated repair, mismatch repair, and DNA synthesis by error-prone polymerases. In this study, we manipulated the number of cell generations by varying the rate at which cultures of a mouse cell line were replenished with fresh medium. We found that the frequency of mutants does not necessarily increase with the number of cell generations. On the contrary, a greater number of divisions can lead to a lower frequency of mutants, indicating that cell division is not a rate-limiting step in the hypermutation process. Thus, when comparing mutation rates, we suggest that rates are more appropriately expressed as mutations per day than per cell generation.

Error-Prone DNA Polymerases iota and beta Are Over-Expressed in B-CLL Cells: Correlation with Specific IgVH Point-Mutations and Implication for the Pathogenesis of Intraclonal IgVH Diversification.

A somatic hypermutation (SHM) process is known to be ongoing in IgVH genes of B-CLL cells (aka intraclonal IgVH diversification) introducing non-random point-mutations due to the combination of two events: a positive selection operated by the antigen and/or the activation of a yet poorly investigated SHM machinery. We analysed here the expression of DNA polymerases (pol) in >95% pure CD19+cells from 44 B-CLLs, all characterized for IgVH mutations (<2%,16 cases; >2%, 28 cases) by cloning and sequencing 5–10 transcripts/B-CLL. The investigated DNA pols were pol alpha, delta, epsilon (proof-reading, involved in DNA replication), pol zeta, eta, iota (error-prone, translesion DNA pols) and pol beta (error-prone, involved in base-excision repair, BER). DNA pol expression, investigated by real-time PCR, was reported as relative levels of DNA pol/beta2microglobulin ratio. Expression of DNA pols was also studied in a series of non-CLL B cell lines and in purified naive (CD19+CD27−) and memory (CD19+CD27+) B cells from PB samples of 5 healthy donors. DNA pols iota and beta were the major DNA pols in B-CLLs; they were expressed at median levels which were from 4.7 (pol beta, range 3.6–6.7, p<0.001) to 19.8 (pol iota, range 15.1–28.2, p<0.0000) folds higher than those of the other DNA pols; these latter were all expressed at significantly lower levels than in normal (naive/memory) B cells or non-CLL B cell lines. As compared to non-CLL B cell lines, DNA pols beta and iota were expressed at 86.5 (p=0.00012) and 7.7 (p<0.0000) folds higher levels, respectively. In addition, pol beta was the sole DNA pol expressed in B-CLL cells at higher (2.5-fold) levels than in normal naive/memory B cells (p=0.028). Although the high expression, DNA pol beta and iota half-lives (57 and 83 mins, respectively), evaluated by blocking RNA synthesis by 5.0 mg/ml actinomycin D up to 4 hrs, were comparable to those of the other DNA pols (range 44–83 mins). By analysing the pattern of specific IgVH mutations in 57 transcripts from 10 B-CLLs with >2% IgVH mutations and lacking antigen-selection, we demonstrated significant correlations (r > 0.70) between pol beta expression and IgVH point-mutations targeting the nucleotide A (A->C, r=0.72), T (T->X, r=0.71; T->C, r=0.82) or C (C->X, r=0.70). Conversely, no correlations were detected in 145 transcripts from 18 B-CLLs with >2% IgVH mutations and evidence of antigen-selection, suggesting that, in these cases, the effects of DNA pols could be masked by the positive pressure exerted by the antigen. In B-CLL cells, DNA pol beta expression was also compared with the expression of activation-induced cytidine deaminase (AID) and uracil-DNA glycosylase-2 (UNG2), enzymes involved in the preliminary steps of SHM. Interestingly, pol beta expression levels were significantly higher (p=0.014) in UNG2+ (14/44), as compared to UNG2- (29/44) cases (median relative levels=16.8 vs. 3.9). A similiar trend was also observed by comparing AID+ with AID- B-CLLs (median relative levels=11.2 vs. 6.8). Taken together, these findings suggest a non-canonical SHM process being active in B-CLL cells in which AID/UNG2 create the abasic sites, while the error-prone DNA pol beta (and iota) may be responsible of specific IgVH point-mutations mainly through a process (BER) not necessary implying DNA replication.

Aberrant Somatic Hypermutation Targets an Extensive Set of Genes in Diffuse Large B-Cell Lymphoma.

Genomic instability is a driving force in tumor development that can be achieved by a variety of mechanisms, such as defective chromosome segregation or inactivation of the DNA mismatch repair pathway. Although B-cell lymphomas are associated with chromosomal translocations deregulating oncogene expression, a mechanism for genome-wide instability during lymphomagenesis has long not been described. We have reported that the somatic hypermutation process (SHM), which normally targets the immunoglobulin variable region (IgV) and BCL6 genes in germinal center (GC) B-cells, functions aberrantly in >50% of diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma (Pasqualucci et al., Nature 412:341, 2001). As a consequence, multiple somatic mutations are introduced into the 5′ region of genes that do not represent physiologic SHM targets, including known proto-oncogenes such as PIM1, PAX5, RhoH/TTF and cMYC. To further define the extent of this phenomenon, termed aberrant somatic hypermutation (ASHM), and to identify additional hypermutated loci of possible pathogenetic significance in DLBCL, we screened 113 genes for the presence of mutations affecting their 5′ sequences (≥1.3 Kb from the transcription start site, the target region for SHM) in 10 DLBCL cell lines. Fifteen genes (13.3%) were found to harbor a significant number of mutations (p<0.05), with 70% of the cell lines being mutated in 7 or more genes; among these, six B-cell specific loci -BCL7A, CIITA, IRF4, LRMP, NCOA3 and SIAT1- carried 9–53 mutational events distributed in 20 to 70% of the cases, corresponding to an overall mutation frequency of 0.032–0.15% (frequency in the mutated cases: 0.07–0.25%). The same genes were found hypermutated in a panel of 20 primary DLBCL biopsies, which displayed an overall mutation load of 7 to 45 distinct events/gene (total N=125). Mutations were of somatic origin, independent of chromosomal translocations to the Ig loci and were restricted to the first 1.5–2 Kb from the promoter. In addition, analogous to previously identified SHM and ASHM targets, the mutations exhibited characteristic features, including a bias for transitions over transversions, preferential hotspot (RGYW/WRCY motifs) targeting, and higher frequencies at G:C pairs. However, in contrast to physiologic SHM targets such as IgV and BCL6, none of the 4 newly identified hypermutated genes that have been analyzed so far (BCL7A, CIITA, SIAT1, LRMP) displayed significant levels of mutations in purified normal GC B-cells as well as in other B-cell malignancies. This finding indicates that these genes represent aberrant hypermutation targets resulting from a tumor-associated malfunction, possibly a loss of target specificity of the physiologic SHM process. Considering previous results and the present survey, 17 (13%) out of 130 genes investigated have been found involved in ASHM, suggesting that this aberrant activity may involve an extensive set of target genes in DLBCL. Since the mutations affect both regulatory and coding sequences of the targeted genes, aberrant SHM may represent a major contributor to the pathogenesis of this disease and may explain in part its phenotypic and clinical heterogeneity.

IG Heavy and Light Chain Variable Genes in Chronic Lymphocytic Leukemia Exhibit Distinct Somatic Mutation Patterns and a Comparable Imprint of Antigen Selection.

We conducted a parallel analysis of somatic mutations in paired IG heavy (HC) and light chain (LC) chain genes in a series of 253 (165 κ- and 88 λ-LC expressing) chronic lymphocytic leukemia (CLL) cases. IGHV/IGKV/IGLV sequences were 100% homologous to germline in 32/33/27% of cases; had 97–99.7% homology in 50/27/37,5% of cases; less than 97% homology in 18/40/35% of cases. IGHV sequences were generally more mutated than the corresponding IGKV/IGKL sequences (average mutation load for IGHV/IGKV/IGLV genes: 94.4/96.4/96.8); 18/253 cases (7.1%) had more mutated LCs than HCs. The most frequent genes among 100% homologous sequences were IGHV4-39 and IGHV1-69 (10/16 and 9/15 cases, respectively) and IGKV1-39/1D-39 (9/19 cases); among IGLV genes, 3/13 IGLV3-21 sequences were unmutated and 9/13 had >98% homology. The most frequent genes in the “<97%-homology” subgroup were IGHV3-7 and IGHV4-34 (18/19 and 23/27 cases, respectively), IGKV3-20 (12/25 cases) and IGLV2-8 (8/14 cases). Analysis after the multinomial distribution model disclosed significant evidence for positive selection by classical T-dependent antigen from both chains in 43 cases, from HCs only in 53 cases and from LCs only in 16 cases; 27 HC+LC mutated cases (7/27 with <97% homology) had no evidence for antigen selection from either HCs or LCs. Mutations followed a similar pattern, with elevated replacement/silent (R/S) ratios in CDRs (mean, 2.8, 2.6 and 3.7 for IGHV, IGKV and IGLV, respectively) and decreased R/S in FRs (mean, 1.4, 1.5 and 1.6 for IGHV, IGKV and IGLV, respectively). R mutations in FRs usually led to substitution by aminoacids of similar charge and size. The somatic hypermutation process generally spread diversity to the periphery of the CDR1 region. Mutational “hotspots” differed not only between HCs and LCs but among subgroups (eg, IGHV1 vs. IGHV3 vs. IGHV4: thus, S-92 in IMGT-FR3 was mutated in 52/21/5% of IGHV4/3/1 sequences; IGKV1/3 vs. IGKV2 subgroup genes; IGLV1 vs. IGLV3 subgroup genes) or even individual genes, e.g. IGHV1-8, IGHV3-23, IGHV4-34, IGKV2-30, IGLV3-21; in this context, a subgroup of three mutated IGHV4-34 sequences was identified, all in sIgG+ cases, with similar HCDR3 regions and associated with IGKV2-30 κ-LCs with identical KCDR3; furthermore, in the case of IGLV3-21, an already described association with IGHV3-21 HCs was again noted in our series (three cases). Importantly, some IMGT positions were universal “hotspots” (eg. 32 in IMGT-CDR1) or “coldspots” (both invariant C at IMGT positions 23/104 in all sequences; IMGT positions 41, 43, 74, 75 and 102 for all IGHV subgroups; 16, 41, 76, 87, 89 and 98 for all IGKV subgroups; 41, 43, 50, 70, 76, 83, 89, 98, 99 and 102 for all IGLV subgroups). Finally, IGLV sequences showed a lower incidence of CDR1 and FR2/3 mutations compared to IGKV sequences. In conclusion, a complementary imprint of antigen selection in CLL might be witnessed either by IGHV, IGKV or IGLV rearranged sequences. These results provide further evidence for the role of antigen selection in the pathogenesis of at least a proportion of CLL cases.

Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells.

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POLbeta, POLdelta, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POLbeta, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POLbeta dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A).

Analysis of IgV gene mutations in B cell chronic lymphocytic leukaemia according to antigen-driven selection identifies subgroups with different prognosis and usage of the canonical somatic hypermutation machinery.

Cases of B-cell chronic lymphocytic leukaemia (B-CLL) with mutated (M) IgV(H) genes have a better prognosis than unmutated (UM) cases. We analysed the IgV(H) mutational status of B-CLL according to the features of a canonical somatic hypermutation (SHM) process, correlating this data with survival. In a series of 141 B-CLLs, 124 cases were examined for IgV(H) gene per cent mutations and skewing of replacement/silent mutations in the framework/complementarity-determining regions as evidence of antigen-driven selection; this identified three B-CLL subsets: significantly mutated (sM), with evidence of antigen-driven selection, not significantly mutated (nsM) and UM, without such evidence and IgV(H) gene per cent mutations above or below the 2% cut-off. sM B-CLL patients had longer survival within the good prognosis subgroup that had more than 2% mutations of IgV(H) genes. sM, nsM and UM B-CLL were also characterized for the biased usage of IgV(H) families, intraclonal IgV(H) gene diversification, preference of mutations to target-specific nucleotides or hotspots, and for the expression of enzymes involved in SHM (translesion DNA polymerase zeta and eta and activation-induced cytidine deaminase). These findings indicate the activation of a canonical SHM process in nsM and sM B-CLLs and underscore the role of the antigen in defining the specific clinical and biological features of B-CLL.

The pattern and distribution of immunoglobulin VH gene mutations in chronic lymphocytic leukemia B cells are consistent with the canonical somatic hypermutation process

AbstractThe overexpanded clone in most B-cell-type chronic lymphocytic leukemia (BCLL) patients expresses an immunoglobulin (Ig) heavy chain variable (VH) region gene with some level of mutation. While it is presumed that these mutations were introduced in the progenitor cell of the leukemic clone by the canonical somatic hypermutation (SHM) process, direct evidence of such is lacking. Nucleotide sequences of the Ig VH genes from 172 B-CLL patients were analyzed. Previously described VH gene usage biases were noted. As with canonical SHM, mutations found in B-CLL were more frequent in RGYW hot spots (mutations in an RGYW motif = 44.1%; germ line frequency of RGYW motifs = 25.6%) and favored transitions over transversions (transition-transversion ratio = 1.29). Significantly, transition preference was also noted when only mutations in the wobble position of degenerate codons were considered. Wobble positions are inherently unselected since regardless of change an identical amino acid is encoded; therefore, they represent a window into the nucleotide bias of the mutational mechanism. B-CLL VH mutations concentrated in complementarity-determining region 1 (CDR1) and CDR2, which exhibited higher replacement-to-silent ratios (CDR R/S, 4.60; framework region [FR] R/S, 1.72). These results are consistent with the notion that VH mutations in B-CLL cells result from canonical SHM and select for altered, structurally sound antigen receptors. (Blood. 2004;103:3490-3495)