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PubMed2003

Somatic mutations and ageing in silico.

Considerable evidence points to an accumulation of somatic mutations in older cells and organisms but the causal role of mutations in the ageing process is still unclear. In addition to demonstrating that mutations accumulate, it is important to address the question of whether they do so at a sufficient rate and with a dynamic profile that is consistent with them playing a causative role. We describe the development of in silico models that can be used to explore the role of somatic mutations in ageing and which form a part of a growing effort to build predictive mathematical and computer models that can help unravel the complexity of the functional genomics of ageing. Our models address, in particular, how mutations affect populations of dividing cells like human fibroblasts, in which the challenge to the somatic mutation theory is greatest, since selection at the cellular level will tend to suppress the accumulation of mutations.

Aging↗

PubMed1993

Analysis of somatic mutation activity in multiple V kappa genes involved in the response to 2-phenyl-5-oxazolone.

We have studied somatic mutation activity early in a response to 2-phenyl-5-oxazolone coupled to ovalbumin (phOx-OVA). Although the V kappa Ox1 gene rearranged to J kappa 5 is known to predominate in this response, other closely related V kappa genes are involved. We compared the introduction of point mutations into V kappa Ox1 genes and into a set of related V kappa genes rearranged to the same J kappa segment at two time points after primary immunization. The result showed that quantitation of mutations in a single rearrangement substrate leads to an underestimation of the total mutational activity. There is pronounced somatic mutation activity early within genes that may be absent later in the response. We also show that multiple somatic mutations can be detected in B cells from draining lymph nodes after foot-pad injection with phOx-OVA already at day 7 after immunization. The data suggest a system in which mutation acts early in the response on a wide range of substrates and that selection and expansion of high affinity paratopes occurs later.

Animals↗

PubMed1999

Germline and somatic mutations in exon 15 of the APC gene and K-ras mutations in duodenal adenomas in patients with familial adenomatous polyposis.

BACKGROUND: In sporadic colorectal adenomas mutations in the adenomatous polyposis gene (APC) are among the first gene aberrations to appear. In familial adenomatous polyposis (FAP) the patients already have a germline mutation in the APC gene. To investigate the natural history of duodenal adenomas in FAP patients, we examined germline and somatic mutations of the APC gene and K-ras mutations in these lesions. METHODS: Frozen sections from 54 duodenal polyps from 31 FAP patients were used to histologically verify the presence of adenomatous growth in the mucosa; the rest of each biopsy specimen was processed for DNA extraction. APC exon 15 was investigated with the protein truncation test (PTT), using four overlapping polymerase chain reaction (PCR) fragments, and samples showing an APC mutation were thereafter sequenced. The adenomas were examined for K-ras mutations by use of a combination of the 'enriched PCR method' and temporal temperature gradient electrophoresis. RESULTS: APC germline mutations in exon 15 were found in 19 of 31 (61%) patients, whereas somatic mutations were localized to 12 of 54 (22%) duodenal adenomas. In seven adenomas both the germline and the somatic mutations were found, whereas five small adenomas showed somatic mutations only. There was no tendency for more mutations to be detected in large and severely dysplastic adenomas compared with small and mildly dysplastic ones. K-ras mutations were found in four (7%) duodenal adenomas. CONCLUSIONS: The low rate of somatic APC and K-ras mutations in duodenal adenomas may indicate another neoplastic pathway than in FAP adenomas of the large bowel, or that a modifier gene is cosegregating with the disease.

Adenoma↗

PubMed1995

The effect of carrier and carrier priming on the kinetics and pattern of somatic mutation in the V chi Ox1 gene.

Priming mice with a chicken gamma globulin (CGG) carrier protein significantly accelerated the onset of somatic mutation in the V chi Ox1 gene when the mice were subsequently immunized with 2-phenyl-5-oxazolone (phOx) coupled to CGG. The first mutations were already detected 7 days after immunization, while in the true primary response, they are not apparent until day 10. It was also found that comparing the mutation pattern of V chi Ox1 genes from hybridomas derived after immunization with phOx coupled to different carriers revealed quite distinct patterns of somatic mutation. Analysis of hybridoma sequences from the primary immune response to phOx-ovalbumin showed that the codons for Ser29, Ser31 and Lys45 were hot-spots for somatic mutation. Thus, the frequency and pattern of somatic mutations in the V chi Ox1 gene depends on the available T cell help as well as on the complex structure of the immunizing antigen.

Animals↗

PubMed2004

Somatic mutations in the D-loop and decrease in the copy number of mitochondrial DNA in human hepatocellular carcinoma.

Somatic mutations in mitochondrial DNA (mtDNA) have been detected in many human cancers, including hepatocellular carcinoma (HCC). The D-loop region was found to be a "hot spot" for mutation in mtDNA of the tumors. However, effects of the D-loop mutations on the copy number of mtDNA in tumor tissues are poorly understood. Using direct sequencing, we examined mutations in the D-loop region of mtDNA in 61 HCCs and the corresponding non-tumor liver tissues. The results revealed that 39.3% of the HCCs carried somatic mutation(s) in the D-loop of mtDNA, and most of these mutations were homoplasmic. Moreover, 37.0% (10/27) of these mutations were T-to-C and G-to-A transitions and 40.7% (11/27) of them were located in the polycytidine stretch between nucleotide position (np) 303 and 309 of mtDNA. In addition, we found that mtDNA copy number of HCC was significantly decreased in 60.5% of the patients with hepatoma, especially in those with somatic mutation(s) in the D-loop of mtDNA (17/24). This decrease in mtDNA copy number was highly associated with the occurrence of point mutations near the replication origin of the heavy-strand of mtDNA. Interestingly, we found that 42.9% (6/14) of the HCCs without mutation in the D-loop had a reduced copy number of mtDNA, indicating that other unidentified factors involved in mitochondrial biogenesis might be defective in the tumor. The results obtained in this study strongly suggest that somatic mutations in the D-loop together with the decrease in the copy number of mtDNA may be an important event during the early phase of liver carcinogenesis.

Base Sequence↗

PubMed1995

Somatic mutation of human immunoglobulin V genes in the X-linked HyperIgM syndrome.

Somatic mutation of Ig variable regions occurs prominently in germinal centers, but it has been debated whether the mutation process initiates in germinal centers or is activated before germinal center entry of B cells. We have analyzed for the presence of somatic mutation in Ig gene rearrangements of the nonpolymorphic human VH6 gene in the X-linked HyperIgM syndrome, which is associated with defective CD40 ligand expression and absence of germinal centers and generation of memory B lymphocytes. IgM and rare IgG VH6 productive rearrangements were isolated from PBL of patients with X-linked HyperIgM syndrome. Although the majority of both the IgM and IgG VH6 rearrangements had a germline VH6 sequence, 7 of 102 VH6 IgM and 1 of 6 IgG rearrangements had a mutated VH6 gene. The mutation frequency (mutations/bp) was 1.4% with a range of 2-9 mutations per clone, a mutation frequency lower, however, than that observed in IgM (3.2%) and IgG (5.4%) VH6 rearrangements of normal individuals. These results suggest that somatic mutation may be initiated in a CD40 ligand-independent pathway before entry of B cells into germinal centers, but fails to achieve the high mutation frequency observed in the presence of germinal centers.

Amino Acid Sequence↗

PubMedDate not supplied

Somatic mutations at microsatellite loci in western Redcedar (Thuja plicata: Cupressaceae).

A per-generation somatic mutation rate for microsatellites was estimated in western redcedar (Thuja plicata, Donn ex D. Don.: Cupressaceae). A total of 80 trees representative of the average size and age of reproductive trees were sampled in four natural populations in southwestern British Columbia. Samples of bulked haploid megagametophytes were collected from two or three positions on each tree, assuming that the collections were far enough apart that the same mutant sector was not sampled twice. All samples were genotyped at eight microsatellite loci. A single mutation corresponding to a stepwise increase in one dinucleotide repeat was detected. The estimated mutation rate for microsatellites was 6.3 x 10(-4) mutations per locus per generation (or 3.1 x 10(-4) per allele per generation), with a 95% confidence interval of 3.0 x 10(-5) to 4.0 x 10(-3) mutations per locus. Somatic mutations can contribute to a greater mutational load in trees, as compared to shorter lived plants, and genotypic mosaics within an individual have important implications for plant defense strategies and plant evolution.

Alleles↗

PubMed1995

Defining subsets of naive and memory B cells based on the ability of their progeny to somatically mutate in vitro.

The increased affinity of memory antibody responses is due largely to the generation and selection of memory B cells that accumulate somatic mutations after initial antigenic stimulation. Further affinity maturation and mutation also accompany subsequent immunizations. Previous studies have suggested that, like primary antibody-forming cell (AFC) clones, secondary AFC do not accumulate further mutations and, therefore, the origins of progressive affinity maturation remain controversial. Here, we report the generation of somatically mutated memory B cell clones in vitro. Our findings confirm the existence of a naive B cell subset whose progeny, rather than generating AFC, somatically mutate and respond to subsequent antigenic stimulation. Interestingly, upon stimulation, a subset of memory B cells also generates antigen-responsive cells that accumulate further somatic mutations.

Amino Acid Sequence↗

PubMed2003

Molecular heterogeneity in MCL defined by the use of specific VH genes and the frequency of somatic mutations.

This study explores whether the presence of somatic mutations or a biased use of IgV(H) genes were associated with the clinical features in a series of 96 patients with mantle cell lymphoma (MCL). The cases were studied by seminested polymerase chain reaction using primers from the FR1 and J(H) regions. There was an unexpectedly high frequency of somatic mutations, with 29 of 103 sequences showing more than 2% of mutations. Biased usage of specific V(H) segments was also found; the most widely used genes in this series were V(H)3-21 (10 cases), V(H)3-23 (9 cases), V(H)4-34 (11 cases), and V(H)4-59 (9 cases). V(H) mutation frequency, taking into account different thresholds, did not distinguish different overall survival probabilities. Nevertheless, a more frequent use of V(H)3-21 or V(H)4-59 (8 of 18) was observed in the group of long-term survivors (18 cases > 5 years; P <.01). None of these long-term survivors presented the V(H)3-23 gene rearrangement. As in other lymphoproliferative disorders, the expression of CD38 or p53 or both was associated with a poorer survival probability. This nonrandom usage of IgV(H) segments suggests that specific antigens may play a pathogenically relevant role in the genesis or progression of subsets of MCL cases and may help in distinguishing a significant group of MCL long-term survivors.

Adult↗

PubMed2004

DNA repair gene polymorphisms, pre-natal factors and the frequency of somatic mutations in the glycophorin-A gene among healthy newborns.

This study investigates variation in somatic mutation frequency, as measured by the glycophorin-A (GPA) somatic mutation assay, in relation to polymorphic variation among 435 newborn babies in DNA repair genes XRCC1, XRCC3 and XRCC4 and gender, parental age, social class and smoking habits. The three polymorphisms under investigation were an Arg --> Gln substitution at codon 399 in exon 10 of XRCC1, a Thr --> Met substitution at codon 241 in exon 7 of XRCC3 and an Ile --> Thr substitution at codon 401 in exon 4 of XRCC4. The study population is an extension of that previously analysed for GPA mutations and XRCC1 polymorphisms. A significant difference was seen in the earlier work in the genotype distribution for the XRCC1 Arg399Gln polymorphism between the main population and the small number with extreme values for NN variant frequency and this was maintained in the larger study group (OR 3.20 [95% CI: 1.16, 8.81]) P = 0.043). No such association was seen for XRCC3 or XRCC4 polymorphisms. When adjustments were made for multiple testing, neither N0 nor NN variant frequencies in the main study population were found to be influenced by the polymorphisms in XRCC1, XRCC3, or XRCC4. In addition, neither maternal or paternal smoking, age or social class nor the gender of the offspring were found to affect variant frequencies nor were variant frequencies influenced by any interaction between any of these factors and genotype. It is concluded that the genotypic variation in DNA repair genes examined in this study has no discernable effect on the genesis of the somatic mutations observed at birth.

DNA Repair↗

PubMed1998

Somatic mutation in the neonatal mouse.

Several mechanisms that diversify the adult immune repertoire, such as terminal deoxynucleotidyl transferase-dependent N region addition, are not available to the neonatal mouse. One important process that contributes to protective immunity in the adult is somatic mutation, which plays a major role in the generation of high affinity memory B cells. It is not clear whether B cells in the neonatal mouse can activate the somatic mutation machinery. To investigate this, we immunized neonates with poly(L-Tyr,L-Glu)-poly-D,L-Ala-poly-L-Lys complexed with methylated BSA, or (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma-globulin. Eight to fourteen days after priming, V(D)J rearrangements of known V(H) genes (V(H)SM7 family) were screened for mutations using a temperature-melt hybridization assay and oligonucleotide probes specific for complementarity-determining regions I and II; possible mutations were confirmed by sequence analysis. More mutations per sequence were found in heavy chains from neonates immunized with (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma-globulin than in those from neonates immunized with poly(L-Tyr,L-Glu)-poly-D,L-Ala-poly-L-Lys complexed with methylated BSA. Mutations were found in heavy chains lacking N regions, suggesting that B cells of the putative fetal lineage can somatically mutate and diversify an initially limited repertoire. Since neonates immunized as early as 1 or 2 days after birth had mutations, the somatic mutation machinery can be activated soon after birth, suggesting that early vaccination should result in affinity maturation and protective immunity in the neonate.

Animals↗

PubMed2001

Development of single-chain Fv fragments from a human anti-double-stranded DNA antibody to study the influence of somatic mutations on antigen binding.

OBJECTIVE: The monoclonal IgG anti-double-stranded (ds) DNA antibody 32B9, obtained from a patient with systemic lupus erythematosus, was found to be encoded by somatically mutated immunoglobulin genes. We examined the input of several somatic mutations into antibody specificity and affinity. METHODS: Five single-chain (sc) Fv fragments [variable domain of the heavy chain (V(H))-linker-variable domain of the light chain (V(L))] derived from 32B9 were constructed and expressed in Escherichia coli. These scFv fragments contained V(H) or V(L) fragments, differing in the somatic mutation pattern. The antigen binding features of the 32B9 IgG were compared with the corresponding scFv fragments, and the binding to DNA of all fragments was analyzed by ELISA. Binding constants to dsDNA were determined by surface plasmon resonance and ELISA. RESULTS: The scFv 32B9 reflected the binding features of the 32B9 IgG. Independently of the somatic mutations, all scFv fragments bound to dsDNA in ELISA. The affinity data indicated that the mutations studied had only a marginal effect on affinity maturation of the 32B9. DISCUSSION: We discuss the approach to constructing scFv fragments as a tool to study autoantibody maturation.

Antibodies, Antinuclear↗

PubMed2006

The nucleotide targets of somatic mutation and the role of selection in immunoglobulin heavy chains of a teleost fish.

Sequence analysis of H chain cDNA derived from the spleen of an individual catfish has shown that somatic mutation occurs within both the VH- and JH-encoded regions. Somatic mutation preferentially targets G and C nucleotides with approximately balanced frequencies, resulting in the predominant accumulation of G-to-A and C-to-T substitutions that parallel the activation-induced cytidine deaminase nucleotide exchanges known in mammals. The overall mutation rate of A nucleotides is not significantly different from that expected by sequence-insensitive mutations, and a significant bias exists against mutations occurring in T. Targeting of mutations is dependent upon the sequence of neighboring nucleotides, allowing statistically significant hotspot motifs to be identified. Dinucleotide, trinucleotide, and RGYW analyses showed that mutational targets in catfish are restricted when compared with the spectrum of targets known in mammals. The preferential targets for G and C mutation are the central GC positions in both AGCT and AGCA. The WA motif, recognized as a mammalian hotspot for A mutations, was not a significant target for catfish mutations. The only significant target for A mutations was the terminal position in AGCA. Lastly, comparisons of mutations located in framework region and CDR codons coupled with multinomial distribution studies found no substantial evidence in either independent or clonally related VDJ rearrangements to indicate that somatic mutation coevolved with mechanisms that select B cells based upon nonsynonymous mutations within CDR-encoded regions. These results suggest that the principal role of somatic mutation early in phylogeny was to diversify the repertoire by targeting hotspot motifs preferentially located within CDR-encoded regions.

Amino Acid Motifs↗

PubMed1999

Primary diffuse large B cell lymphoma of the stomach: analysis of somatic mutations in the rearranged immunoglobulin heavy chain variable genes indicates antigen selection.

Gastric low grade MALT lymphomas show a pattern of somatic mutations in their rearranged immunoglobulin genes, indicative of antigen selection. This provides evidence for antigen stimulation in the lymphomagenesis. Gastric diffuse large B cell lymphomas develop secondary to low grade MALT lymphoma or de novo. To study whether antigen-selection is also a feature of primary diffuse large B cell lymphomas, we analysed somatic mutations in the rearranged immunoglobulin heavy chain (IgH) variable genes (VH). The rearranged VH genes of six cases of gastric primary diffuse large B cell lymphoma were amplified from genomic or complementary DNA by a VH gene family-specific polymerase chain reaction method. The PCR products were directly sequenced and were compared to published germline sequences to analyse somatic mutations. Similarly to low grade MALT lymphomas 5/6 primary diffuse large B cell lymphomas show a pattern of somatic mutation in their rearranged VH genes, indicative of antigen selection and suggesting a role for antigens in lymphomagenesis. One case showed bi-allelic VH gene rearrangements, which were non-functional due to extensive deletions. Antigen selection could not be demonstrated or excluded. Antigen selection is a common feature in most analysed primary diffuse large B cell lymphomas, although some heterogeneity in the mechanisms involved in the lymphomagenesis of gastric primary diffuse large B cell lymphomas has not been excluded entirely (case 4).

Aged↗

PubMed1993

Somatic mutations of the APC gene in precancerous lesion of the stomach.

The adenomatous polyposis coli (APC) gene, responsible for familial adenomatous polyposis, is also associated with development of sporadic tumors in digestive system as colon, stomach, or pancreas. In order to investigate whether or not APC mutations occur as an early genetic event during gastric carcinogenesis, we examined somatic mutations of APC in flat adenomas of the stomach. DNAs isolated from flat adenomas were examined by means of an RNase protection analysis coupled with polymerase chain reaction (PCR) followed by DNA sequencing of the PCR products. By screening a mutation cluster region (MCR: codons between 1286 and 1513) of APC in which two-thirds of somatic mutations were detected in colorectal tumors, somatic mutations were found in four of ten flat adenomas: three of which caused truncation of the gene product due to a nonsense mutation or 4-bp deletion; one other was a point mutation that altered amino acid from alanine to threonine. Our results imply that APC plays a crucial role in an early step of gastric carcinogenesis, as was observed in colorectal carcinogenesis.

Adenoma↗

PubMed2004

Frequent somatic mutations of mitochondrial DNA in esophageal squamous cell carcinoma.

Recent studies of various cancers, such as those of the breast, head and neck, bladder and lung, reported that 46-64% of somatic mutations in the D-loop region of mitochondrial DNA (mtDNA) are observed. However, in esophageal cancer, only a low rate (5%) of somatic mutations has so far been reported in one article (Hibi, K. et al., Int J Cancer 2001;92:319-321). Thus, to confirm this we analyzed the somatic mutations for hypervariable regions (HVR-I and HVR-II) in the D-loop of mtDNA to reevaluate the possibility of mitochondrial genetic instability in this cancer. We amplified both HVRs by PCR and DNA samples obtained from 38 esophageal tumors and matched normal tissues, and then sequenced them. Comparing the sequences of tumors to those of normal tissues, we found 14 somatic mutations in 13 patients (34.2%). Eleven mutations were at the C consecutive stretch from position 303 to 309 of MITOMAP in the mitochondria databank (http://www.mitomap.org/), 1 at position 215 in HVR-II and 2 at positions 16,304 and 16,324 in HVR-I. There were 41 types of germ line variations in HVR-I including 2 not so far recorded in the mtDNA databank and 17 in HVR-II including 1 not yet recorded. We also determined nuclear genome instability of these 38 specimens by analyzing 3 independent microsatellite sequences. While 4 specimens showed a single microsatellite change, which is tumor specific, we did not find any co-relation between a somatic mtDNA mutation and microsatellite instability of nuclear genome DNA. These results suggest that mtDNA mutations might show a genetic instability in esophageal cancer independently from a nuclear genome instability.

Age Distribution↗

PubMed1991

DNA fingerprinting: the utilization of minisatellite probes to detect a somatic mutation in the proteus syndrome.

Syndromes with localized or segmental abnormalities have been proposed to be the result of a somatic mutation leading to the presence of somatic mosaicism in the tissue. The Proteus syndrome, with its hemihypertrophy, macrodactyly and exostoses, has features which would indicate that the phenotype results from such events. The success of utilizing DNA fingerprint probes to detect somatic mutations in cancer raised the possibility that a similar approach might be successful in an investigation of two patients with the Proteus syndrome. Single band differences were detected with the probe 33.6 in a pair of monozygotic twins discordant for Proteus and in a comparison of tissue from normal and affected areas in another patient. These findings would appear to confirm the hypothesis that the Proteus syndrome results from a somatic mutation. Furthermore, the results indicate that DNA fingerprinting may offer a valuable technique for identifying probes for investigations of similar syndromes.

Abnormalities, Multiple↗

PubMed1995

Use of transgenic mouse models for studying somatic mutations in aging.

Theories on the causes of aging, based on the accumulation of somatic mutations in tissues of an organism, were formulated decades ago, but remain insufficiently tested. Transgenic animals, equipped with integrated bacterial reporter genes that can be efficiently rescued from total genomic DNA of all tissues and organs, represent ideal tools for investigating the types and frequencies of spontaneous mutants accumulating during aging. The first of such systems, based on the transgenic integration of bacteriophage lambda shuttle vectors that contain the bacterial lacZ gene as mutational target, was constructed in our laboratory and is now routinely used. Results obtained with this and the related LacI system that are relevant for the somatic mutation theory of aging will be discussed. One conclusion is that, due to the nature of the transgene, lambda-based systems have the disadvantage that deletion type mutations are underrepresented in comparison to point mutations. To overcome those limitations, we constructed a new transgenic mouse model carrying a pUR288 plasmid shuttle vector with the lacZ reporter gene. Some preliminary data obtained with this model serve to illustrate its potential use to extensively test the somatic mutation theory of aging.

Aging↗