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PubMed2004

PKD1 intron 21: triplex DNA formation and effect on replication.

Although autosomal dominant polycystic kidney disease is transmitted in an autosomal dominant fashion, there is evidence that the pathophysiology of cystogenesis involves a second hit somatic mutation superimposed upon the inherited germline mutation within the renal tubule cells. The polypurine.polypyrimidine (Pu.Py) tract of PKD1 intron 21 may play a role in promoting somatic mutations. To better characterize this tract and to evaluate its potential to participate in mutagenesis, we investigated the thermodynamics of intramolecular triplex formation by 15 Pu.Py mirror repeat tracts from PKD1 intron 21 by 2D gel electrophoresis. We demonstrate that intramolecular triplexes form with modest superhelical tensions for all the tracts examined. Primer extension studies demonstrated significant polymerase arrest within the Pu.Py tracts in one direction of replication only. We found correlation between polymerization arrest and both the potential length of the triplex and superhelical tension of intramolecular triplex formation. The presence of a Pu.Py tract also led to a replication blockade and double-strand breakage using an SV40 in vitro replication assay with HeLa cell extracts. During DNA replication, the G-rich template of the PKD1 Pu.Py tracts may form a triplex structure with the nascent strand, thereby blocking replication and potentially leading to recombination and mutation.

DNA↗
PubMed2002

Analysis of B cell memory formation using DNA microarrays.

DNA microarray analysis of B cell subsets has identified comprehensive programs of gene expression that distinguish B cells at discrete stages of differentiation. The next task is to identify key genetic signals within these complex programs that regulate the dynamic cellular events during B cell activation in vivo. After stimulation with antigen, naïve B cells proliferate and differentiate, and then produce antibodies. Crucial qualitative differences in antibody responses are observed depending on whether or not B cells receive T cell help during activation. Proteins, lipopolysaccharides, and polysaccharides stimulate T-dependent (TD), T-independent type 1 (TI-1), and type 2 (TI-2) antibody responses, respectively. Only TD responses generate somatically mutated antibody-forming (plasma) cells and memory B cells, which produce high affinity anamnestic responses to subsequent antigen challenge. Somatic mutation of immunoglobulin genes occurs during B cell proliferation in germinal centres (GC), which are typical in TD responses but rare in TI responses. However, we have described a model, which is exceptional because numerous large GC form in response to a model TI-2 antigen, (4-hydoxy-3-nitrophenyl) acetyl (NP)-Ficoll. Significantly, these GC undergo involution before memory B cells are generated. This model provides an opportunity to investigate the genetic signals that drive memory cell formation, and we have compared global gene expression in TI and TD GC to identify a relatively small number of genes that are differentially expressed between the two prototypic B cell responses. This model demonstrates how genome-scale technology can be adapted to investigate specific aspects of B cell biology.

Animals↗
PubMed1999

Alterations of the DR5/TRAIL receptor 2 gene in non-small cell lung cancers.

Chromosome 8p21-22 is a frequent site of allelic deletions in many types of human tumors, including non-small cell lung cancer (NSCLC). Tumor necrosis factor-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2) is a cell-surface receptor involved in cell death signaling. The TRAIL-R2 gene recently has been mapped to chromosome 8p21-22. To explore the possibility that the TRAIL-R2 gene might be the relevant gene to the frequent deletion of 8p21-22 in NSCLC, we have analyzed the entire coding region and all splice sites of TRAIL-R2 for the detection of the somatic mutations in a series of 104 NSCLCs. Overall, 11 tumors (10.6%) were found to have TRAIL-R2 gene mutations in the death domain known to be involved in the transduction of an apoptotic signal. Our data indicate that somatic mutation of TRAIL-R2 may play a role in the pathogenesis of some NSCLCs and that the TRAIL-R2 gene is one of the genes relevant to the frequent loss of chromosome 8p21-22 in NSCLC.

Adenocarcinoma↗
PubMed1990

Modulation of pro-epidermal growth factor, pro-transforming growth factor alpha and epidermal growth factor receptor gene expression in human renal carcinomas.

We have analyzed the expression of the genes for the precursors of epidermal growth factor (pro-EGF) and transforming growth factor alpha (proTGF-alpha) as well as for the EGF receptor in tissue specimens of a large number of adult patients with renal cell carcinoma. Since normal kidney tissue was available from the same patients we could directly compare the expression of these genes in tumors with that in adjacent normal renal tissue. Our experiments reveal underexpression of the proEGF gene in all tumors analyzed (21 of 21) and overexpression of the genes for proTGF-alpha (33 of 33 analyzed) and EGF receptor (22 of 23 analyzed) in tumor samples, when compared with normal kidney tissue. The expression of the proTGF-alpha gene appeared to depend on grade and differentiation of the tumor, since well differentiated tumors (grade 1) expressed more proTGF-alpha mRNA than the adjacent normal tissue but significantly less than poorly differentiated tumors (grade 2 or 3), which are the most aggressive ones. In none of these tissue specimens did we find, by Southern analysis, amplification of the proTGF-alpha or EGF receptor gene. Therefore, overexpression of these genes must be due to another effect, perhaps an alteration of their mRNA turnover. Although the EGF receptor gene (c-erbB1) is overexpressed in nearly all carcinomas analyzed, there was no linear coexpression with the proTGF-alpha gene. In contrast, transcription of the proEGF gene was completely turned off in tumor tissue. Although we have found by restriction fragment length polymorphism analysis, in one of three tumor samples, evidence for a somatic mutation within the proEGF gene, we do not know yet, due to the limited number of Southern analyses, whether this somatic mutation is causally involved in the decrease of proEGF mRNA expression and, hence, is representative of renal cell carcinoma. To our knowledge, this is the first observation on primary tumor tissue in humans that upon malignant transformation the gene for a polypeptide growth factor gene is underexpressed.

Blotting, Northern↗
PubMed2001

Autosomal dominant polycystic kidney disease: modification of disease progression.

Autosomal dominant polycystic kidney disease is a common inherited disorder, which is characterised by the formation of fluid-filled cysts in both kidneys that leads to progressive renal failure. Mutations in two genes, PKD1 and PKD2, are associated with the disorder. We describe the various factors that cause variation in disease progression between patients. These include whether the patient has a germline mutation in the PKD1 or in the PKD2 gene, and the nature of the mutation. Detection of mutations in PKD1 is complicated, but the total number identified is rising and will enable genotype-to-phenotype studies. Another factor affecting disease progression is the occurrence of somatic mutations in PKD genes. Furthermore, modifying genes might directly affect the function of polycystins by affecting the rate of somatic mutations or the rate of protein interactions, or they might affect cystogenesis itself or clinical factors associated with disease progression. Finally, environmental factors that speed up or slow down progress towards chronic renal failure have been identified in rodents.

Animals↗
PubMed1990

Pathogenic anti-DNA antibodies in SLE: idiotypic families and genetic origins.

We have adopted an idiotypic approach to study the double stranded DNA (dsDNA) binding antibodies of systemic lupus erythematosus (SLE). Three anti-idiotypic reagents, 8.12, 3I, and F4, identify cross reactive idiotypes that are each expressed on anti-dsDNA antibodies in the sera of many patients with SLE. These idiotypic antibodies are implicated in the pathogenesis of SLE as they are present in immune complex deposits in the kidneys of patients with SLE glomerulonephritis. The autoantibody associated idiotypes are also expressed on antibodies that do not bind DNA. We are investigating the origin of the pathogenic anti-dsDNA antibodies of SLE by comparing the autoantibodies, the antibodies to foreign antigens, and the myeloma proteins that express each SLE associated idiotype. In conjunction with serological analysis of these idiotypic systems, molecular genetic studies indicate that both the 8.12 and the 3I autoantibody associated idiotypes may be germline encoded, while the F4 idiotype is generated by somatic mutation. The data further suggest that the antigenic specificity of the pathogenic anti-DNA antibodies of SLE is acquired through somatic mutation of germline immunoglobulin genes. By studying the regulation of genes capable of encoding pathogenic autoantibodies, in both SLE patients and non-autoimmune individuals, we may be able to elucidate the pathogenesis of autoimmune disease and begin to design more effective therapeutic interventions.

Antibodies, Antinuclear↗
PubMed1996

Low incidence of BRCA2 mutations in breast carcinoma and other cancers.

Inherited mutant alleles of familial tumour suppressor genes predispose individuals to particular types of cancer. In addition to an involvement in inherited susceptibility to cancer, these tumour suppressor genes are targets for somatic mutations in sporadic cancers of the same type found in the familial forms. An exception is BRCA1, which contributes to a significant fraction of familial breast and ovarian cancer, but undergoes mutation at very low rates in sporadic breast and ovarian cancers. This finding suggests that other genes may be the principal targets for somatic mutation in breast carcinoma. A second, recently identified familial breast cancer gene, BRCA2 (refs 5-8), accounts for a proportion of breast cancer roughly equal to BRCA1. Like BRCA1, BRCA2 behaves as a dominantly inherited tumour suppressor gene. Individuals who inherit one mutant allele are at increased risk for breast cancer, and the tumours they develop lose the wild-type allele by heterozygous deletion. The BRCA2 coding sequence is huge, composed of 26 exons that span 10,443 bp. Here we investigate the rate of BRCA2 mutation in sporadic breast cancers and in a set of cell lines that represent twelve other tumour types. Surprisingly, mutations in BRCA2 are infrequent in cancers including breast carcinoma. However, a probable germline mutation in a pancreatic tumour cell line suggests a role for BRCA2 in susceptibility to pancreatic cancer.

BRCA2 Protein↗
PubMedDate not supplied

Variation of spontaneous and induced mitotic recombination in different Drosophila populations: a pilot study on the effects of polyaromatic hydrocarbons in six newly constructed tester strains.

A set of six Drosophila strains was developed, by inducing by chemical treatment with N-ethyl-N-nitrosourea (ENU) new white and, in some strains, yellow mutations in 3 wild-type (WT) and 3 insecticide-resistant (IR) populations. These strains were previously shown to vary with regard to contents and inducibility of microsomal oxidative enzymes (Zijlstra et al., 1984). In this pilot study results from a first evaluation of these strains in somatic mutation experiments are reported, using as genotoxins an aromatic amine (2-naphthylamine, 2-NA), one substituted (9,10-dimethylanthracene, DA) and one non-substituted (benzo[a]pyrene, BP) polycyclic aromatic hydrocarbon. Developing larvae heterozygous for white were chronically exposed to three different exposure doses of each carcinogen. Adult females were inspected for the occurrence of mosaic light clones in their eyes, using the somatic mutation and recombination test (SMART). Evidence is presented indicating strong genotype-dependent variation in both spontaneous and chemically induced mutational and recombinational events in somatic cells of Drosophila. The spontaneous frequencies varied from 3.5% (Hikone-R), 4.3% (Berlin-K), 6.3% (Oregon-K), 9.1% (91-C), 20.5% (Haag-79) to 49.1% (91-R), corresponding to a 14-fold difference in spot frequencies between the two extremes. BP, DA and 2-NA were readily detectable in both Hikone-R (IR) and Oregon-K (WT), less so in 91-C (WT) and Haag-79 (IR), whereas the performance of strain Berlin-K (WT) was rather poor. The special problem with strain 91-R was the high frequency with which mosaic light spots occur not only in female genotypes heterozygous for white, but also in homozygous condition in the original stock. The up to 20-fold variation in induced spot frequencies between different genotypes poses questions for further investigations with respect to the genetic constitution of the various strains and the role of enzyme induction on somatic cell mutagenicity, which in this system is predominantly the result of mitotic recombination.

2-Naphthylamine↗
PubMed2001

A comparative analysis of the immunological evolution of antibody 28B4.

In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K(d) = 25 microM) and 28B4 Fab (K(d) = 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95(H)Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. (1998) Science 279, 1929]. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed.

Amino Acid Sequence↗
PubMed2004

Mutational analysis of susceptibility genes RNASEL/HPC1, ELAC2/HPC2, and MSR1 in sporadic prostate cancer.

Three putative prostate cancer-susceptibility genes, RNASEL/HPC1 at 1q24, MSR1 at 8p22, and ELAC2/HPC2 at 17p11, have recently been identified. Our objective was to investigate somatic mutations in these genes in sporadic prostate cancer. We analyzed 39 clinical prostate cancer specimens, 10 prostate cancer xenografts (LuCaP series), and 4 prostate cancer cell lines (LNCaP, DU145, PC-3, and MPC-3) for genetic changes using denaturing high-performance liquid chromatography and direct sequencing in order to screen the whole coding regions of RNASEL and MSR1, as well as exons 7 and 17 of ELAC2. The known 471delAAAG truncating mutation was found in the RNASEL gene in cell line LNCaP. The only new missense variation in RNASEL, Gly296Val, was found in cell line DU145, but not in any other samples. RNASEL and ELAC2 also showed the common missense polymorphic changes. A previously reported truncating mutation (Arg293X) was found in MSR1 in the germ line of one individual. Our results indicate that inactivation of the RNASEL, ELAC2, or MSR1 genes by somatic mutation is a rare phenomenon in sporadic prostate cancer.

Animals↗
PubMed1994

Loss of heterozygosity of MCC is not associated with mutation of the retained allele in sporadic colorectal cancer.

The adenomatous polyposis coli (APC) gene, which transmits familial adenomatous polyposis, is frequently mutated in sporadic colorectal tumours. Acquired somatic mutations have also been reported in a second gene, mutated in colorectal cancer (MCC), which lies within 500 kb of APC on chromosome 5q21 and has thus been implicated in tumour development. Further evidence for an oncosuppressor gene other than APC on chromosome 5q comes from recent studies of lung, renal and hepatic cancers in which there is loss of heterozygosity of 5q21 but no somatic APC mutations. To investigate the relative importance of APC and MCC in sporadic colorectal cancer, we have assessed the extent of 5q21 allelic loss in 80 carcinomas. All informative tumours exhibiting allelic loss had deletions which included both APC and MCC. In 21 tumours with loss of heterozygosity in MCC we have screened the entire coding region of the gene for mutation of the retained allele and found no evidence for mutation. The data indicate that independent loss of MCC is a rare event, and that in cases where allele loss occurs mutation of the retained allele is uncommon. This suggests that MCC does not function as an independent tumour suppressor in the majority of colorectal cancers.

Alleles↗
PubMed1996

Immune response to different doses of a hapten of fluorescein isothiocyanate analyzed by two-dimensional affinity electrophoresis.

The immune response to different doses of a hapten of fluorescein isothiocyanate (FITC) in BALB/c mice was analyzed by two-dimensional affinity electrophoresis (2-D AEP). The mice were immunized with different doses (0.5 microgram, 5 micrograms, 50 micrograms, 500 micrograms and 2.5 mg) of FITC-conjugated bovine serum albumin (BSA). The antibodies to FITC bovine serum albumin (BSA) were separated into a large number of IgG spots due to differences in their isoelectric points (pI) and binding affinity to FITC ligand immobilized in the gel. The IgG spots, showing identical affinity to the ligand but different pI, have been considered as an IgG family. The affinity and quantity of IgG families changed with the increase in FITC-BSA dosage. With a low dose (5 micrograms) most of the families showed high affinity (Kd < 1 microM < Kd < 50 microM) and low affinity (Kd > 50 microM) antibodies were produced. The increase of FITC-BSA up to 500 micrograms markedly increased the quantity of IgG spots showing a variety of affinity to FITC. However, 2.5 mg FITC-BSA did not increase the quantity and heterogeneity of IgG spots significantly. The changes in the heterogeneity and quantity of anti-FITC antibodies and the subclass switch were observed over the course of immunization. The heterogeneity and the quantity of IgG1, IgG2b and IgG3 antibodies increased markedly during the first and the second immunization, whereas an increase in the heterogeneity and the quantity of IgG2a antibody was observed in the third immunization. This suggests that the subclass switch to IgG1, IgG2b and IgG3 and the somatic mutation of IgG1, IgG2b and IgG3 occur during the first and the second immunization, but the subclass switch to IgG2a and the somatic mutation of IgG2a seem to occur later than that of the other IgG subclasses.

Animals↗
PubMed2006

The epidermal growth factor receptor gene sequence is highly conserved in primary gastric cancers.

BACKGROUND AND OBJECTIVES: Recent studies have disclosed the presence of somatic mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancers (NSCLC), and susceptibility to the EGFR tyrosine kinase inhibitor (gefitinib) was determined by the presence of mutations in the kinase domain of this gene. We thus predicted a clinical benefit of gefitinib against the 12% of primary colorectal cancers exhibiting a mutation reflective of this potential distinctive susceptibility. PATIENTS AND METHODS: The mutation status of the kinase domain in EGFR in different primary cancers has important clinical consequences, because the presence of a mutation is recognized as a reliable indicator for the effectiveness of gefitinib administration. In the current study, we investigated the presence of somatic mutations in exons 18-21 coding the ATP-binding domain in five gastric cancer cell lines and 39 primary gastric cancers and their corresponding normal tissues. RESULTS AND CONCLUSIONS: The kinase domain of EGFR is highly conserved in whole gastric cancer cell lines and cases, therefore treatment with gefitinib is unfortunately not recommended for such malignancy.

Adult↗
PubMed1992

Antimutagenic effects in humans.

The application of antimutagenicity studies to human somatic mutation is discussed, with emphasis on the potential for future studies. Five assay-gene combinations are now available for measuring human somatic mutation in lymphocytes and erythrocytes. Results with these combinations have defined the human background levels, and show clear responses of mutant frequency to a variety of mutagens. The testing of antimutagenic effects on background frequencies is feasible, but has not yet been done. The major uncertainty in such studies is the unknown age of mutant cells in the background, since only the newly forming mutants are potentially susceptible to most antimutagenic treatments. Intervention studies in the face of active mutagenicity and the use of other genotoxicity endpoints, such as chromosome aberrations, micronuclei and DNA adducts, are considered briefly.

Antimutagenic Agents↗
PubMed1989

Molecular dissection of an antigen-specific immune response.

Selection defines the repertoire of the primary response, and also which cells differentiate and enter the pool of the memory cells. Whereas the primary response is drawn from the germline repertoire, the memory response consists overwhelmingly of cells which have been hypermutated and selected to express receptors of high affinity. The accumulation of somatic mutations over time indicates that the activation of the memory cell by antigen also reactivates the hypermutation mechanism. Since the frequency of somatic mutations correlates with an increase in antibody affinity, an efficient selection operates not only on the primary B cells when first stimulated by antigen, but also on the memory cells after secondary or tertiary immunization.

Animals↗
PubMed1996

Contribution of heavy chain junctional amino acid diversity to antibody affinity among p-azophenylarsonate-specific antibodies.

We showed previously that heavy chain gene junctional amino acid differences among unmutated p-azophenylarsonate (Ars) Abs that share a unique gene segment combination encoding these V regions, termed "canonical," alter affinity. To determine the contribution of junctional amino acid differences to binding, we introduced, by site-directed mutagenesis, various amino acids at position 100 and/or 107 (sequential numbering) into the unmutated Ab 36-65. Among 22 mutant Abs, 15 preserved or showed increased Ars binding (1-to 12.9-fold increase) relative to Ab 36-65, while 7 Abs exhibited lower affinity (< or = 0.5-fold). As much as a 150-fold difference in Ars binding was observed between 2 Abs with different sets of junctions (Asn100/Tyr107 and Val100/Lys107). Thus, amino acid replacements at D gene junctions can produce changes in affinity greater than those for any V region somatic mutation observed thus far in vivo among anti-Ars Abs and, potentially, can result in preferential selection of Abs containing certain junctions during affinity maturation. We combined five different junctional residue pairs with mutations at H chain positions 58 and 59 that are known to be recurrent in vivo and are associated with increased Ars affinity. The mutant Abs all showed increased affinity, indicating that despite variation in D gene junctions of Ars-binding canonical Abs, the combined mutations are additive for enhancement of Ars affinity. These additive effects reflect the "adaptability" of the canonical gene segment combination in sustaining somatic mutations leading to affinity maturation.

Amino Acid Sequence↗
PubMed1998

Heterogeneity of microsatellite mutations within and between loci, and implications for human demographic histories.

Microsatellites have been widely used to reconstruct human evolution. However, the efficient use of these markers relies on information regarding the process producing the observed variation. Here, we present a novel approach to the locus-by-locus characterization of this process. By analyzing somatic mutations in cancer patients, we estimated the distributions of mutation size for each of 20 loci. The same loci were then typed in three ethnically diverse population samples. The generalized stepwise mutation model was used to test the predicted relationship between population and mutation parameters under two demographic scenarios: constant population size and rapid expansion. The agreement between the observed and expected relationship between population and mutation parameters, even when the latter are estimated in cancer patients, confirms that somatic mutations may be useful for investigating the process underlying population variation. Estimated distributions of mutation size differ substantially amongst loci, and mutations of more than one repeat unit are common. A new statistic, the normalized population variance, is introduced for multilocus estimation of demographic parameters, and for testing demographic scenarios. The observed population variation is not consistent with a constant population size. Time estimates of the putative population expansion are in agreement with those obtained by other methods.

Adenocarcinoma↗