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PubMed1994

Variable region gene analysis of B cell subsets derived from a 4-year-old child: somatically mutated memory B cells accumulate in the peripheral blood already at young age.

Tonsillar germinal center and immunoglobulin M+ (IgM+)IgD+ B cells as well as peripheral blood (PB) CD5+ and CD5- (conventional) B cells from a 4-yr-old child were isolated and nucleotide sequences of expressed Ig heavy chain variable regions encoded by VH4 gene family members were determined from amplified cDNA. Whereas both tonsillar IgM+IgD+ cells and the majority of IgM-expressing CD5+ and CD5- PB B cells showed no or little somatic mutation, tonsillar germinal center (GC) B cells and IgG-expressing PB B cells carried a high load of somatic mutations in their V region genes. This suggests that somatically mutated memory B cells which have switched isotype accumulate in the PB already at young age. Their frequency seems to increase with age. On the other hand, the antibody repertoire of tonsillar IgM+IgD+ B cells and the majority of IgM-expressing PB B cells is determined by germline-encoded specificities and by generation of variability in the complementary determining region III through VH-DH-JH recombination. A fraction of IgM-bearing PB B cells carries somatically mutated V region genes and probably represents GC-derived B cells which have left the GC at an early stage of the GC reaction without undergoing isotype switching. 10 VH4 germline genes were found to be expressed. Three gene segments were overrepresented in the sequence collection (35 of 50 clones): VH4.21 (30%), V71-4 (20%), and 3D279D (20%). It appears that most potentially functional VH4 germline genes are expressed in peripheral B cells. Some members of this VH gene family are clearly overrepresented over others.

Amino Acid Sequence↗
PubMed2003

Somatic mutation analysis of p53 and ST7 tumor suppressor genes in gastric carcinoma by DHPLC.

AIM: To verify the effectiveness of denaturing high-performance liquid chromatography (DHPLC) in detecting somatic mutation of p53 gene in gastric carcinoma tissues. The superiority of this method has been proved in the detection of germline mutations, but it was not very affirmative with respect to somatic mutations in tumor specimens. ST7 gene, a candidate tumor suppressor gene identified recently at human chromosome 7q31.1, was also detected because LOH at this site has also been widely reported in stomach cancer. METHODS: DNA was extracted from 39 cases of surgical gastric carcinoma specimen and their correspondent normal mucosa. Seven fragments spanning the 11 exons were used to detect the mutation of p53 gene and the four exons reported to have mutations in ST7 gene were amplified by PCR and directly analyzed by DHPLC without mixing with wild-type allele. RESULTS: In the analysis of p53 gene mutation, 9 aberrant DHPLC chromatographies were found in tumor tissues, while their normal-adjacent counterparts running in parallel showed a normal shape. Subsequent sequencing revealed nine sequence variations, 1 polymorphism and 8 mutations including 3 mutations not reported before. The mutation rate of p53 gene (21%) was consistent with that previously reported. Furthermore, no additional aberrant chromatography was found when wild-type DNA was added into the DNA of other 30 tumor samples that showed normal shapes previously. The positivity of p53 mutations was significantly higher in intestinal-type carcinomas (40%) than that in diffuse-type (8.33%) carcinomas of the stomach. No mutation of ST7 gene was found. CONCLUSION: DHPLC is a very convenient method for the detection of somatic mutations in gastric carcinoma. The amount of wild type alleles supplied by the non-tumorous cells in gastric tumor specimens is enough to form heteroduplex with mutant alleles for DHPLC detection. ST7 gene may not be the target gene of inactivation at 7q31 site in gastric carcinoma.

Base Sequence↗
PubMed2000

Antigen persistence is required for somatic mutation and affinity maturation of immunoglobulin.

Whether germinal centers (GC) with follicular dendritic cell (FDC) clusters are the essential sites for affinity maturation of immunoglobulin is still controversial. To re-evaluate the role of GC / FDC in affinity maturation and somatic mutation in a defined antigen system, lymphotoxin-alpha(- / -) and TNF receptor I(- / -) mice, lacking GC / FDC, were immunized with (4-hydroxy-3-nitrophenyl) acetyl-sheep RBC (NP-SRBC). In contrast to soluble hapten-carrier systems, NP-SRBC allows us to compare affinity maturation in the presence or absence of adjuvant. These mice showed a dramatically impaired ability to generate high-affinity IgG to NP, but retained the ability to produce low-affinity anti-NP IgG when NP-SRBC was used in the absence of adjuvant. In contrast to wild-type mice, somatic mutation of the expressed IgG heavy chain gene was rarely detected in these GC / FDC-deficient mice. This suggests that GC / FDC are essential for affinity maturation. Trapping antigen-specific B cells inside the T cell zone of TNFRI(- / -) mice may prolong the interaction between T and B cells, which allows class switching but no further affinity maturation of IgG. Interestingly, GC / FDC-deficient mice could be induced to generate high-affinity, somatically mutated IgG antibodies by immunization with the same amount of NP-SRBC antigen emulsified in incomplete Freund's adjuvant or repeated immunization with the antigen alone. Thus, these data support a model in which prolonged availability of antigen is required for somatic mutation and affinity maturation, and FDC or adjuvants facilitate such processes by slowly releasing antigens.

Animals↗
PubMed2001

Germline and somatic mutations in hMSH6 and hMSH3 in gastrointestinal cancers of the microsatellite mutator phenotype.

Hereditary and sporadic gastrointestinal cancer of the microsatellite mutator phenotype (MMP) is characterized by a remarkable genomic instability at simple repeated sequences. The genomic instability is often caused by germline and somatic mutations in DNA mismatch repair (MMR) genes hMSH2 and hMLH1. The MMP can be also caused by epigenetic inactivation of hMLH1. The MMP generates many somatic frameshift mutations in genes containing mononucleotide repeats. We previously reported that in MMP tumors the hMSH6 and hMSH3 MMR genes often carry frameshift mutations in their (C)(8) and (A)(8) tracks, respectively. We proposed that these 'secondary mutator mutations' contribute to a gradual manifestation of the MMP. Here we report the detection of other frameshift, nonsense, and missense mutations in these genes in colon and gastric cancers of the MMP. A germline frameshift mutation was found in hMSH6 in a colon tumor harboring another somatic frameshift mutation. Several germline sequence variants and somatic missense mutations at conserved residues were detected in hMSH6 and only one was detected in hMSH3. Of the three hMSH6 germline variants in conserved residues, one coexisted with a somatic mutation at the (C)(8) track and another had a somatic missense mutation. We suggest that some of these germline and somatic missense variants are pathogenic. While biallelic hMSH6 and hMSH3 frameshift mutations were found in some tumors, many tumors seemed to contain only monoallelic mutations. In some tumors, these somatic monoallelic frameshift mutations at the (C)(8) and (A)(8) tracks were found to coexist with other somatic mutations in the other allele, supporting their functionality during tumorigenesis. However, the low incidence of these additional somatic mutations in hMSH6 and hMSH3 leaves many tumors with only monoallelic mutations. The impact of the frameshift mutations in gene expression was studied by comparative analysis of RNA and protein expression in different tumor cell clones with different genotypes. The results show that the hMSH6 (C)(8) frameshift mutation abolishes protein expression, ruling out a dominant negative effect by a truncated protein. We suggest the functionality of these secondary monoallelic mutator mutations in the context of an accumulative haploinsufficiency model.

Amino Acid Sequence↗
PubMed1990

Clustered H chain somatic mutations shared by anti-p-azophenylarsonate antibodies confer enhanced affinity and ablate the cross-reactive idiotype.

A cluster of four consecutive CDR2 somatic mutations are shared by the VH regions of two independently isolated hybridoma antibodies with specificity for p-azophenylarsonate (Ars). The mutations appear to be derived by a series of independent events. To assess the influence of these shared somatic mutations on antibody affinity for Ars and on idiotypy, we introduced them, via site-directed mutagenesis, into the V region of an anti-Ars antibody that was otherwise unmutated and we eliminated them from the mutated context of one of the two antibodies in which they were originally found. Results of affinity measurements by fluorescence quenching and of idiotypic binding assays performed on these engineered mutants demonstrated that the shared mutations increased affinity for Ars and eliminated the predominant Id associated with strain A anti-Ars antibodies and four of five idiotypes defined by anti-idiotypic mAb. These results support the interpretation that a strong affinity-based selection pressure has favored the clonal expansion of B cells with receptors containing these mutations despite the loss of a predominant Id. Thus, in producing antibodies containing V regions conferring high affinity for Ag, the combined processes of somatic mutation and clonal selection have generated a common structural solution through parallel repeats.

Amino Acid Sequence↗
PubMed1997

Molecular single-cell analysis reveals that CD5-positive peripheral blood B cells in healthy humans are characterized by rearranged Vkappa genes lacking somatic mutation.

B cells expressing the CD5 cell surface antigen are involved in certain B cell malignancies and autoimmune diseases. From studies in the mouse, it emerged that CD5+ B cells represent a separate lineage of B lymphocytes that, in contrast to conventional (CD5-) B cells, are not driven into T cell-dependent immune responses in which rearranged variable (V) region genes are diversified by somatic hypermutation. Against this background it came as a surprise that human disease-involved CD5-positive autoreactive B cells as well as B cell chronic lymphocytic leukemias can harbor somatically mutated V region genes. Recent V gene analyses on CD5+ B cells in healthy adults did not give rise to a clear picture about the fraction of somatically mutated among all CD5+ B cells. In this work we used a molecular single-cell analysis to determine reliably the frequency of mutated CD5+ B cells in healthy humans: single, kappa light chain-expressing CD5+ peripheral blood B cells were isolated by flow cytometry, and rearranged Vkappa genes were amplified by PCR. From one donor, CD5+CD19+ B cells were analyzed. Since CD5+ B cells were found among IgM+IgD+ and IgM+IgD- cells (but almost not among class-switched cells) from two other donors, individual cells corresponding to these IgM-expressing subsets were investigated separately. The sequence analysis of rearranged Vkappa genes revealed that most if not all CD5+ B cells in healthy humans carry unmutated V region genes. From one of the donors, a novel polymorphic Jkappa2 gene segment was identified. To explain the discrepancy between the frequent occurrence of disease-associated somatically mutated CD5+ B cells and the low incidence or absence of somatic mutation in normal CD5+ B cells, we speculate that CD5+ B cells usually do not participate in germinal center reactions, but if they occasionally do so, they may be at an increased risk to become involved in autoimmune diseases or B cell malignancies.

Adult↗
PubMed2006

Signs of positive selection of somatic mutations in human cancers detected by EST sequence analysis.

BACKGROUND: Carcinogenesis typically involves multiple somatic mutations in caretaker (DNA repair) and gatekeeper (tumor suppressors and oncogenes) genes. Analysis of mutation spectra of the tumor suppressor that is most commonly mutated in human cancers, p53, unexpectedly suggested that somatic evolution of the p53 gene during tumorigenesis is dominated by positive selection for gain of function. This conclusion is supported by accumulating experimental evidence of evolution of new functions of p53 in tumors. These findings prompted a genome-wide analysis of possible positive selection during tumor evolution. METHODS: A comprehensive analysis of probable somatic mutations in the sequences of Expressed Sequence Tags (ESTs) from malignant tumors and normal tissues was performed in order to access the prevalence of positive selection in cancer evolution. For each EST, the numbers of synonymous and non-synonymous substitutions were calculated. In order to identify genes with a signature of positive selection in cancers, these numbers were compared to: i) expected numbers and ii) the numbers for the respective genes in the ESTs from normal tissues. RESULTS: We identified 112 genes with a signature of positive selection in cancers, i.e., a significantly elevated ratio of non-synonymous to synonymous substitutions, in tumors as compared to 37 such genes in an approximately equal-sized EST collection from normal tissues. A substantial fraction of the tumor-specific positive-selection candidates have experimentally demonstrated or strongly predicted links to cancer. CONCLUSION: The results of EST analysis should be interpreted with extreme caution given the noise introduced by sequencing errors and undetected polymorphisms. Furthermore, an inherent limitation of EST analysis is that multiple mutations amenable to statistical analysis can be detected only in relatively highly expressed genes. Nevertheless, the present results suggest that positive selection might affect a substantial number of genes during tumorigenic somatic evolution.

Amino Acid Sequence↗
PubMed1997

Somatic mutation in VH complementarity-determining region 2 and framework region 2: differential effects on antigen binding and Ig secretion.

The extent to which somatic mutation impairs the Ig complementarity-determining region (CDR) and framework region (FRW) structure/function is not clear. Previously, we found that the VH CDR2 of the murine T15 Ab is highly sensitive to mutation; 56% (26 of 46) of Abs mutated in vitro had reduced or no Ag binding capability, and 9% were secretion impaired. Here we test whether the T15 VH CDR2 structure is unique by mutating the VH CDR2 of the anti-PC-protein murine Ab, PCG1-1. PCG1-1 VH is encoded by the M141 gene and is unrelated in sequence or structure to that of T15 VH1. The majority (54%, 20 of 37) of PCG1-1 mutants carrying one to five mutations in VH CDR2 had reduced or abolished Ag binding, while 10% were secretion impaired. Taken together, mutational analysis of the VH1 and VH M141 genes demonstrates that impaired binding and secretion may be common outcomes of CDR2 somatic mutation. We also tested the tolerance of the VH FRW2 of T15 to mutation, expecting this sequence-conserved region to be highly sensitive to alterations. However, FRW2 accommodated many nonconservative changes, and only 12% (3 of 25) of secreted mutants had impaired Ag binding. Moreover, mutations in FRW2 caused secretion defects in 24% (8 of 33), a frequency twice that of VH CDR2 mutants. A total of 16 unique secretion mutants have now been identified. These findings suggest that B cell losses from somatic mutation may be extensive and due to varied causes not all related to Ag binding.

Animals↗
PubMed1997

Somatic mutation theory, DNA repair rates, and the molecular epidemiology of p53 mutations.

The theory of somatic mutagenesis predicts that the frequency pattern of induced selectable mutations along a gene is the product of the probability patterns of the several sequential steps of mutagenesis, e.g., damage, repair, polymerase misreading, and selection. Together, the variance of these component steps is propagated to generate a mutagen's induced mutational spectrum along a gene. The step with the greatest component of variance will drive most of the variability of the mutation frequency along a gene. This most variable step, for UV-induced mutations, is the cyclobutyl pyrimidine dimer repair rate. The repair rate of cyclopyrimidine dimers is quite variable from nucleotide position to nucleotide position and we show that this variation along the p53 gene drives the C-->T transition frequency of non-melanocytic skin tumors. On showing that the kinetics of cyclopyrimidine dimer repair at any one nucleotide position are first order, we use this kinetic and the somatic mutation theory to derive Leq, the adduct frequency along a gene as presented to a DNA polymerase after a cell population reaches damage-repair equilibrium from a chronic dose of mutagen. Leq is the product of the first two sequential steps of mutagenesis, damage and repair, and the frequency of this product is experimentally mapped using ligation-mediated PCR. The concept of Leq is applied to mutagenesis theory, chronic dose genetic toxicology, genome evolution, and the practical problems of molecular epidemiology.

Animals↗
PubMed2006

Somatic mutations of epidermal growth factor receptor in bile duct and gallbladder carcinoma.

OBJECTIVE: Conventional therapies are still unsuccessful in patients with carcinoma arising from the biliary tract. Somatic mutations of the epidermal growth factor receptor (EGFR) gene and the activation of its downstream pathways predict the sensitivity to small-molecule inhibitors in non-small cell lung carcinoma. Therefore, we analyzed EGFR mutations and related pathways in gallbladder and bile duct carcinomas to consider the possible application of these alternative therapeutic strategies. EXPERIMENTAL DESIGN: Forty paraffin-embedded samples, including intrahepatic or extrahepatic cholangiocarcinoma and gallbladder carcinoma, were studied after tumor cell isolation by laser microdissection and sequencing of EGFR tyrosine kinase domain (exons 18-21). Activation of EGFR pathway was studied by evaluating phosphorylation of mitogen-activated protein kinase and Akt. RESULTS: None of the 40 specimens had mutations in exon 18; one had one missense point mutation in exon 19, two in exon 20, and three in exon 21. In addition, 36 of 40 specimens had the same silent mutation at codon 787 in exon 20, which was also found in peripheral blood cells from healthy donors. Tumor samples harboring EGFR mutation had phosphorylation of one or both downstream transducers analyzed. CONCLUSIONS: This is the first evidence of somatic mutations of the EGFR gene in bile duct carcinoma. Our findings suggest that a subgroup of patients with cholangiocarcinoma or gallbladder carcinoma exhibits somatic mutations of EGFR in the tyrosine kinase domain that can elicit cell signals sustaining survival and proliferation. These tumors might be further evaluated for their susceptibility to small-molecule inhibitor treatment.

Adult↗
PubMed1996

Risk estimation from somatic mutation assays.

The ability to quantify somatic mutations in vivo provides a new source of toxicological information that is relevant to the assessment of cancer risk. The major experimental factors that influence the mutant frequency are age, time after treatment, treatment protocol, and tissue analyzed. In untreated mice, the mutant frequency increases very rapidly with age from conception to birth, more slowly from birth to adulthood, and very slowly thereafter. All somatic tissues studied so far in adults have similar mutant frequencies. The time after treatment (expression time) is the most important experimental variable. The minimum time for expression varies from one tissue to another. To be valid, comparisons between tissues and treatments must be made after complete expression of the mutations. Unfortunately, the minimum expression time has not been characterized in most tissues. Since carcinogens are tissue specific, and many chemicals are distributed in the body in complex patterns, it is to be expected that there will be differences in the frequency of mutation induced in different tissues. As yet this has not been extensively studied. Since the mutations detected by the transgenic assays are neutral, the mutants should accumulate as the integral of the mutation rate. Hence chronic treatment protocols should be more effective than acute and subacute protocols whenever they permit substantially larger doses to be delivered. Such protocols are more relevant to human exposure and are preferable for dose extrapolations. The importance of transcription in determining mutation rates is not yet known, but it is noteworthy that the transgenes are not transcribed whereas the Ioci involved in carcinogenesis are. The mutation spectrum is important for quantitative risk estimation. Risk estimation must also take into account the spectrum of mutations that are involved in the carcinogenic process in the tissue and the spectrum of mutations that are detectable by the assay. New assays are being used to quantify mutations in vivo in order to understand the carcinogenic process, to search for the environmental factors involved in human cancer, and to evaluate the carcinogenic hazard qualitatively.

Animals↗
PubMed2002

Identification of somatic mutations of the RNF6 gene in human esophageal squamous cell carcinoma.

We mapped a tumor suppressor gene locus to an 800-kb interval on human chromosome 13q12.11 for esophageal squamous cell carcinoma (ESCC). Two genes, ML-1 and RNF6, are located within this 800-kb interval. We analyzed both genes for the presence of mutations in 24 ESCC primary tumors and 16 tumor cell lines by directly sequencing the PCR products that were amplified from each exon. No mutation was detected in ML-1. In contrast, three somatic mutations in the RNF6 gene were detected in the ESCC primary tumors, and one mutation was also found in a tumor cell line. Identification of multiple somatic mutations in RNF6 suggests that RNF6 is a potential tumor suppressor gene involved in the pathogenesis of ESCC.

Carcinoma, Squamous Cell↗
PubMed2000

Somatic mutations and genetic polymorphisms of the PPP1R3 gene in patients with several types of cancers.

Recently, we found nonsense and missense mutations of the PPP1R3 (protein phosphatase 1, regulatory subunit 3) gene in diverse human cancer cell lines and primary lung carcinomas, indicating that PPP1R3 functions as a tumor suppressor in human carcinogenesis. In this study, to assess the prevalence of PPP1R3 mutations in human primary cancers and the genetic diversity of the PPP1R3 gene in the human population, somatic mutations and genetic polymorphisms in the PPP1R3 gene were examined in 137 pairs of cancerous and non-cancerous tissues of patients with cancers of colon, ovary, and liver. Five somatic mutations including two missense mutations were detected in three cancerous tissues consisting of two colorectal carcinomas and one ovarian carcinoma. Five novel single nucleotide polymorphisms (SNPs) associated with the substitution of amino acids were also identified in cancer patients, in addition to five known nonsynonymous SNPs, including three previously reported ones as having an impact on the susceptibility to insulin resistant disorders. Differences in the activities and properties of multiple PPP1R3 proteins, which are produced in human cells due to variable somatic mutations and genetic polymorphisms in the PPP1R3 gene, can be involved in human carcinogenesis and susceptibility to diseases.

Amino Acid Substitution↗
PubMed1995

Human anti-Gal heavy chain genes. Preferential use of VH3 and the presence of somatic mutations.

Anti-Gal is the most abundant natural Ab known in humans. It interacts specifically with the carbohydrate structure Gal alpha 1-3 Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope), constitutes approximately 1% of circulating Ig, and is found in all three isotypes in the serum. Anti-Gal is produced in Old World monkeys, apes, and humans, and in no other mammalian species. The objective of this study was to determine the VH genes involved in the synthesis of anti-Gal. B lymphocytes from various individuals were transformed by EBV, the clones producing anti-Gal were isolated, the specificity and affinity of the Abs were determined, and the VH genes were sequenced. The affinity of anti-Gal clones for the free radiolabeled alpha-galactosyl epitope ranged between 1.1 x 10(6) M-1 and 5 x 10(8) M-1. Eight of the nine clones studied used VH3 family genes and one clone used a VH1 family gene. Four of the five VH3 genes used were found to form a cluster of related sequences, suggesting that functional constraints may lead to the use of VH3 genes with structural motifs that are suited for specific interactions with the alpha-galactosyl epitope. Comparison with known germline sequences for all of the clones studied and analysis of autologous germ-line genes in two of the clones indicate that anti-Gal VH genes undergo somatic mutations. These somatic mutations may provide a pool of variants that are available for affinity maturation.

Amino Acid Sequence↗
PubMed1995

Somatic mutations in the BRCA1 gene in sporadic ovarian tumours.

The BRCA1 gene on chromosome 17q21 is responsible for an autosomal dominant syndrome of increased susceptibility to breast and ovarian cancer but no somatic mutations in tumours have yet been described. To study the potential role of BRCA1 in sporadic carcinogenesis, we analysed the genomic DNA of tumour and normal fractions of 47 ovarian cancers for mutations in BRCA1 using the single-strand conformation polymorphism technique. We now describe somatic mutations in the DNA of four tumours which also had loss of heterozygosity (LOH) at a BRCA1 intragenic marker. Our data support a tumour suppressor mechanism for BRCA1; somatic mutations and LOH may result in inactivation of BRCA1 in at least a small number of ovarian cancers.

Adolescent↗
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↗