SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Attention is drawn to the possibility of half chromatid and early somatic mutations and to several implications of these mosaic-yielding events. There is suggestive evidence that spontaneous mutations can result in mosaics. A world-wide cooperative study of Lesch-Nyhan families could determine the extent of half chromatid mutation transmission and early somatic mutation in humans.
Although a small fraction of human cancers have increased rates of somatic mutation because of known deficiencies in DNA repair, little is known about the prevalence of somatic alterations in the vast majority of human cancers. To systematically assess nonsynonymous somatic alterations in colorectal neoplasia, we used DNA sequencing to analyze approximately 3.2 Mb of coding tumor DNA comprising 1,811 exons from 470 genes. In total, we identified only three distinct somatic mutations, comprising two missense changes and one 14-bp deletion, each in a different gene. The accumulation of approximately one nonsynonymous somatic change per Mb of tumor DNA is consistent with a rate of mutation in tumor cells that is similar to that of normal cells. These data suggest that most sporadic colorectal cancers do not display a mutator phenotype at the nucleotide level. They also have significant implications for the interpretation of somatic mutations in candidate tumor-suppressor genes.
The mitochondrial theory of aging proposes that reactive oxygen species (ROS) generated inside the cell will lead, with time, to increasing amounts of oxidative damage to various cell components. The main site for ROS production is the respiratory chain inside the mitochondria and accumulation of mtDNA mutations, and impaired respiratory chain function have been associated with degenerative diseases and aging. The theory predicts that impaired respiratory chain function will augment ROS production and thereby increase the rate of mtDNA mutation accumulation, which, in turn, will further compromise respiratory chain function. Previously, we reported that mice expressing an error-prone version of the catalytic subunit of mtDNA polymerase accumulate a substantial burden of somatic mtDNA mutations, associated with premature aging phenotypes and reduced lifespan. Here we show that these mtDNA mutator mice accumulate mtDNA mutations in an approximately linear manner. The amount of ROS produced was normal, and no increased sensitivity to oxidative stress-induced cell death was observed in mouse embryonic fibroblasts from mtDNA mutator mice, despite the presence of a severe respiratory chain dysfunction. Expression levels of antioxidant defense enzymes, protein carbonylation levels, and aconitase enzyme activity measurements indicated no or only minor oxidative stress in tissues from mtDNA mutator mice. The premature aging phenotypes in mtDNA mutator mice are thus not generated by a vicious cycle of massively increased oxidative stress accompanied by exponential accumulation of mtDNA mutations. We propose instead that respiratory chain dysfunction per se is the primary inducer of premature aging in mtDNA mutator mice.
Leiomyomas are the most common gynecologic tumors in women, but very little is known about their molecular pathology. We used single-stranded conformational polymorphism/heteroduplex analysis to analyze 42 unselected uterine leiomyomas for somatic mutations in all coding exons of the gene encoding CCAAT displacement protein (CDP), as well as exons 5-8 of TP53 and codons 1-36 and 38-80 of KRAS. No somatic mutations were identified in either TP53 or KRAS, indicating that disregulation of these genes is not required for leiomyomas development. Aberrant band shifts were identified in CDP, but these were all germline nonpathogenic variants that have been reported previously. There is good functional and genetic evidence indicating that CDP is a leiomyoma suppressor, but our data suggested that somatic mutations in this gene were rare in unselected uterine leiomyomas. It is possible that CDP belongs to a class of tumor suppressor in which loss of only one copy of the gene, either by genetic or epigenetic mechanisms, is sufficient to allow tumor growth.
Papillary thyroid carcinoma (PTC) is a rare extracolonic manifestation of familial adenomatous polyposis, determined by germline mutations of the adenomatous polyposis coli (APC) gene. The aim of this study was to assess the presence of loss of heterozygosity of APC in the thyroid tumoral tissue. Specimens from six female patients, aged 20-36, were analyzed for germline and somatic mutations of the APC gene by restriction enzyme analysis and sequence analysis. Five of the six also had analysis for ret/PTC, a chimeric gene, the activation of which is restricted to papillary TC. Because a previous study showed that germline mutations in familial adenomatous polyposis-associated thyroid carcinoma were located between codons 140 and 1513, the search for somatic mutations of the APC gene was restricted to this genomic area. Three of the six patients, belonging to the same kindred, had a germline mutation at codon 1061. The remaining three, one per kindred, had germline mutations at codons 1061, 1061, and 1309, respectively. None of the six patients had loss of heterozygosity for APC or somatic mutation in the explored genomic area (codon 545 and codons 1061-1678). Four of five had activation of ret/PTC in the thyroid tumoral tissue, as ret/PTC1 isoform. Either APC has a tissue-specific dominant effect in the thyroid gland or the germline mutation confers a generic susceptibility to cancer development, but other factors (sex-related factors, environmental radiation, modifier genes) are also required for TC development. This usually involves ret/PTC activation, suggesting a possible cooperation between altered function of APC and gain of function of ret.
The mitochondrial (mt) DNA control region (CR) of dogs and wolves contains an array of imperfect 10 bp tandem repeats. This region was studied for 14 domestic dogs representing the four major phylogenetic groups of nonrepetitive CR and for 5 wolves. Three repeat types were found among these individuals, distributed so that different sequences of the repeat types were formed in different molecules. This enabled a detailed study of the arrays and of the mutation events that they undergo. Extensive heteroplasmy was observed in all individuals; 85 different array types were found in one individual, and the total number of types was estimated at 384. Among unrelated individuals, no identical molecules were found, indicating a high rate of evolution of the region. By performing a pedigree analysis, array types which had been inherited from mother to offspring and array types which were the result of somatic mutations, respectively, could be identified, showing that about 20% of the molecules within an individual had somatic mutations. By direct pairwise comparison of the mutated and the original array types, the physiognomy of the inserted or deleted elements (indels) and the approximate positions of the mutations could be determined. All mutations could be explained by replication slippage or point mutations. The majority of the indels were 1-5 repeats long, but deletions of up to 17 repeats were found. Mutations were found in all parts of the arrays, but at a higher frequency in the 5' end. Furthermore, the inherited array types within the mother-offspring pair were aligned and compared so that germ line mutations could be studied. The pattern of the germ line mutations was approximately the same as that of the somatic mutations.
Skin fibroblasts of patients with Cockayne syndrome (CS) are hypersensitive to the lethal or mutagenic effect of ultraviolet light, which may cause genetic instability. Up to now, however, no systematic study of in vivo somatic cell mutation in CS cells has been reported. This article describes our investigation of the mutation frequencies (Mfs) at three different loci, i.e. hypoxanthine-guanine phosphoribosyl transferase (HPRT), T-cell antigen receptor (TCR) and glycophorin A (GPA), in six patients with CS. Mfs at the HPRT and TCR loci were found to be within the normal range as determined in age-matched controls. In the GPA locus of two patients, there was a slight increase, but it was much smaller than that reported in other DNA repair deficient syndromes. The frequency of spontaneous HPRT mutation in Epstein-Barr virus transformed B-lymphoblastoid cells derived from CS patients was similar to that in cells from normal children. The molecular characterization of the representative HPRT mutant T cell clones from CS patients did not show any structural alterations. These results may explain, at least in part, why CS is not associated with predisposition to cancer.
More than half of the anti-Sm hybridomas isolated from MRL/Mp-lpr/lpr (MRL/lpr) mice produce Abs that also bind ssDNA, and half of these bind dsDNA. Intraclonal comparisons indicate that DNA is a selecting Ag for at least some dual-binding clones. To determine whether Sm itself is a selecting Ag for anti-Sm, we have identified the somatic mutations within the expressed VH and V kappa genes of eight anti-Sm hybridomas, six of which do not bind DNA. We find these V genes have between 0 and 12 somatic mutations each, and that four hybridomas possess a higher number of heavy or light chain CDR replacement (R) mutations than expected by chance, suggesting that these anti-Sm-producing B cells have undergone Ag selection. To demonstrate directly the effect of somatic mutation on Sm binding, we have engineered the unmutated counterpart of Ab 2-12, an Sm-specific hybridoma Ab with a nonrandom distribution of V kappa CDR R mutations, and compared its ability to bind Sm and ssDNA with that of the originally isolated 2-12 Ab. We find that the unmutated Ab has a much lower avidity for Sm than the mutant, but, unlike the mutant, it binds ssDNA. We conclude that Sm can drive clonal expansion in the anti-Sm response, and that Sm-only binding B cells can arise from Sm/DNA dual-binding B cell clonal precursors. These data also suggest that dual binding is not necessary to sustain clonal expansion. Thus, this response is unique in that it can be driven by either of two Ags.
Experimental hypofunction of thyroid gland, caused by mercazolil administration, and acute external exposure to 0.5 and 1.0 Gy of gamma-irradiation were shown to result in disturbance of reproductive function in puberal females and of estrogenreceptor interaction, in reduction of pregnancy probability, increase in pre- and postimplantation lethality of fetuses as well as in rise in the frequency of structural mutations in somatic cells of pregnant rats and their offspring.
Familial gastrointestinal stromal tumor (GIST) is a rare autosomal dominant genetic disorder associated with KIT germline mutations. In sporadic forms of the disease, somatic mutations target either KIT or PDGFRA genes. In a kindred in which 5 individuals had GIST, no germline mutation in KIT coding sequence has been detected. We hypothesized that the PDGFRA gene could be a predisposing gene in familial GIST. We sequenced PDGFRA exons 12 and 18 because several somatic mutations were identified within this region. We detected a germline PDGFRA missense mutation, 2675G > T, resulting in a tyrosine substitution for the highly conserved aspartic acid at codon 846. This mutation showed perfect cosegregation with the GIST phenotype among the 7 family members tested. Interestingly, PDGFRA Asp846 is homologous to codon 820, which is located in the KIT tyrosine kinase II domain. In a previous study, a KIT germline Asp820Tyr mutation was detected in a Japanese kindred in which 6 individuals had GIST. Transfection of a KIT820Tyr complementary DNA in nude mice was found to be tumorigenic confirming the oncogenic potential of this mutation. The present study shows that PDGFRA is a second familial GIST predisposing gene. These results indicate a further example of involvement of structurally related genes in familial cancer syndromes.
The VH gene (Variable gene segments of the heavy chain locus) repertoire can be investigated by DNA analysis of rearranged immunoglobulin VH genes, which also allows for an indirect estimation of antibody selection by analysis of somatic mutations. Using a polymerase chain reaction (PCR) it is also possible to analyse these genes in small numbers of cells or even single cells. This approach was chosen to investigate germinal centre like lymphocyte follicles in the synovial membranes of two patients with rheumatoid arthritis (RA) in order to analyse the local humoral immune response in RA. Individual B-cell aggregates of synovial membrane of two patients with RA were isolated by micromanipulation from microscopic slides. VH-DH-JH (variable, diversity, and joining segments of the heavy chain locus) rearrangements in all possible VH-JH combinations were amplified from these B cell foci, cloned and subjected to sequence analysis. Sequence analysis revealed that most of the rearranged VH genes were somatically mutated with at least 1% (range 1.3-14.9%) somatic mutations and therefore were derived from antigen-selected memory B cells. Intraclonal diversity in one-third of the clones indicated the generation of memory B cells in the synovial membrane and characterized the synovial membrane as lymphatic tissue where secondary immune responses to an as yet unknown antigen take place.
It is a common notion that mature B lymphocytes express either kappa or lambda light (L) chains, although the mechanism that leads to such isotypic exclusion is still debated. We have investigated the extent of L chain isotypic exclusion in normal human peripheral blood B lymphocytes. By three-color staining with anti-CD19, anti-kappa, and anti-lambda antibodies we could estimate that 0.2-0.5% of peripheral blood B cells from healthy adults express both kappa and lambda on the cell surface. The kappa+lambda+ cells were sorted, immortalized by Epstein-Barr virus, and five independent clones were characterized in detail. All clones express both kappa and lambda on the cell surface and produce immunoglobulin M that contain both kappa and lambda chains in the same molecule, i.e., hybrid antibodies. Sequencing of the L chains revealed in three out of five clones evidence for somatic mutations. It is interesting to note that among a panel of single receptor B cell clones we identified two lambda+ clones that carried a productively rearranged kappa, which was inactivated by a stop codon generated by somatic mutation. These findings indicate that dual receptor B lymphocytes can be found among mature antigen-selected B cells and suggest that somatic mutation can contribute to increase the degree of isotypic exclusion by inactivating a passenger, nonselected L chain.
Pituitary adenoma is a common neoplasm accounting for 10% of all intracranial tumors. Although the molecular mechanisms underlying the formation of these tumors are largely unknown, a small portion of pituitary adenomas occur in patients with the multiple endocrine neoplasia syndrome type 1 (MEN 1). Although two groups in the United States and Canada have recently reported that sporadic pituitary adenomas very rarely harbor a somatic mutation in the MEN1, MEN1 gene mutation analysis in sporadic pituitary adenomas has not yet been carried out in the Japanese population. To elucidate the potential etiological role of the MEN1 gene in the formation of sporadic pituitary adenomas in Japan, we investigated 40 Japanese patients with sporadic pituitary adenomas (16 hormone-secreting and 24 nonsecreting tumors) for MEN1 gene mutation. Polymerase chain reaction-single-stranded DNA conformation polymorphism analysis and sequencing demonstrated a somatic mutation in the MEN1 gene in only one of the 40 tumors, a prolactinoma, which had a 1-bp deletion in the coding sequence of exon 2. The data suggest that somatic mutations in the MEN1 gene do not play a prominent role in the pathogenesis of sporadic pituitary adenomas.
To examine the possible effects of space radiation on living organisms, fruit flies Drosophila melanogaster were loaded on the US Space Shuttle Endeavour, and after the flight we have analyzed two types of mutations, sex-linked recessive lethal mutations induced in male reproductive cells and somatic mutations which give rise to morphological changes in hairs growing on the surface of wing epidermal cells. Wild type strains and a radiation-sensitive strain mei-41 were used. The frequencies of sex-linked recessive lethal mutations in flight groups were 2 and 3 times higher for wild type Canton-S and mei-41 strains, respectively, than those in ground control groups. By contrast, the frequencies of wing-hair somatic mutations differed little between flight and control groups. The possibility that the space environment causes mutations in certain types of cells such as male reproductive cells, is discussed.
Near-ultraviolet light (UVA: 320-400 nm) constitutes a major part of sunlight UV. It is important to know the effect of UVA on the biological activities of organisms on the earth. We have previously reported that black light induces somatic-cell mutation in Drosophila larvae. To investigate which wavelength of the UVA is responsible for the mutation we have now carried out a series of monochromatic irradiations (310, 320, 330, 340, 360, 380 and 400 nm) on Drosophila larvae, using the large spectrograph of the National Institute for Basic Biology (Okazaki National Research Institutes, Okazaki, Japan). Mutagenic activity was examined by the Drosophila wing-spot test in which we observe mutant wing hair colonies (spots) on the wings of adult flies obtained from the treated larvae. The induction of mutation was highest by irradiation at 310 nm and decreased as the wavelength became longer. Neither the 380 nor the 400 nm light was mutagenic. Excision repair is known to protect cells from UV damage. In the excision-repair-deficient Drosophila, the mutagenic response induced by 310 nm irradiation was 24-fold higher than that of the wild-type (7.2 +/- 1.5 spots/wing/kJ vs 0.3 +/- 0.08 spots/wing/kJ), and at 320 nm the difference of the response was 14-fold (0.21 vs 0.015 +/- 0.005). In the case of irradiation at 330 and 340 nm the difference of the response was only two-fold (at 330 nm, 6.9 +/- 2.9 x 10(-3) vs 3.1 +/- 1.1 x 10(-3) spots/wing/kJ; at 340 nm, 3.5 +/- 0.9 x 10(-3) vs 2.0 +/- 0.7 x 10(-3). These results suggest that the lesion caused in the larvae by 320 nm irradiation may be similar to the damage induced by 310 nm and that the lights of 330 and 340 nm may induce damage different from the lesions induced by shorter-wavelength lights.
While somatic antibody mutants are rare in the preimmune repertoire and in primary immune responses, they dominate secondary and hyperimmune responses. We present evidence that somatic hypermutation is restricted to a particular pathway of B-cell differentiation in which distinct sets of B-cell clones are driven into the memory compartment. In accord with earlier results of McKean et al. (1984) and Rudikoff et al. (1984), somatic mutation occurs stepwise in the course of clonal expansion, before and after isotype switch, presumably at a rate close to 1 X 10(-3) per base pair per generation. At this rate, both selectable and unselectable mutations accumulate in the rearranged V region genes. The distribution of replacement mutations in the V regions shows that a fraction of the mutations in CDRs is positively selected whereas replacement mutations are counterselected in the FRs. By constructing an antibody mutant through site-specific mutagenesis we show that a point mutation in CDR1 of the heavy chain, found in most secondary anti-NP antibodies, is sufficient to increase NP binding affinity to the level typical for the secondary response. Somatic mutation may contribute to the immune repertoire in a more general sense than merely the diversification of a specific response. We have evidence that clones producing antibodies which no longer bind the immunizing antigen can be kept in the system and remain available for stimulation by a different antigen. Somatic mutations are 10 times less frequent in DJH loci than in either expressed or non-expressed rearranged VDJH or VJ loci. We therefore conclude that a V gene has to be brought into the proximity of the DJH segment in order to fully activate the hypermutational mechanism in these loci.
Gastric carcinoma is one of the most common types of cancer in Taiwan. Somatic mitochondrial DNA (mtDNA) alteration in gastric carcinoma and its association with clinicopathologic features remain unclear. When we used polymerase chain reaction (PCR) and direct sequencing, 15 of the 31 (48%) gastric carcinomas displayed somatic mutations in the D-loop region, a hot spot for mutations in mtDNA of human cancers. Ten (67%) cancers with the somatic mutations in the D-loop had insertion or deletion mutations in nucleotide position (np) 303-309 in the mononucleotide repeat region. One carcinoma carried tandem duplication and triplication flanked by mononucleotide repeats starting at np 311 and 568, respectively, in the D-loop. We also detected the common 4,977-bp deletion in 17 (55%) of the noncancerous tissue samples, but only in three (9%) carcinomas. Moreover, we quantified the mtDNA content using a competitive PCR technique and found that mtDNA depletion occurred in 17 (55%) of the gastric carcinomas. Although no significant association was found between clinicopathologic features and the mtDNA mutations in the D-loop, mtDNA depletion was observed significantly in the ulcerated, infiltrating (Borrmann's type III) and diffusely thick (Borrmann's type IV) types of gastric carcinomas (P = 0.018). Our results suggest that somatic mtDNA mutations and mtDNA depletion occur in gastric cancer and that mtDNA depletion is involved in carcinogenesis and/or cancer progression of gastric carcinoma.
von Hippel-Lindau (VHL) disease is a pleiotropic disorder featuring a variety of malignant and benign tumors of the eye, central nervous system, kidney, and adrenal gland. Recently the VHL gene has been identified in the chromosomal region 3p25-26. Prognosis and successful management of VHL patients and their descendants depend on unambiguous diagnosis. Due to recurrent hemangioblastomas, a29-year-old patient without familial history of VHL disease was diagnosed to be at risk for the disease. Histopathological examination of a small renal mass identified a clear cell tumor with a G1 grading. Genetic characterization of the germline and of the renal tumor was performed. Polymerase chain reaction/single strand conformation polymorphism (PCR/SSCP) analysis with primers from the VHL gene identified a deletion of a single nucleotide in exon 2 in the patient's germline and in the tumor, but not in the DNA of his parents. This deletion therefore must be a de novo mutation. Comparative genome hybridization (CGH) and fluorescence in situ hybridization (FISH) analysis of the G1 tumor with differentially labelled yeast artifical chromosome (YAC) clones showed loss of 3p and of the 3p26 signals, respectively. In conclusion, we identified a de novo germline mutation in the VHL gene of a young patient and a somatic chromosome 3p loss at the homologous chromosome 3 in his renal tumor. Our results suggest a recessive mode of inactivation of the VHL gene, providing solid evidence for its tumor-suppressor gene characteristics. Our data show the diagnostic potential of genetic testing, especially in patients without VHL family history. Furthermore, the findings of homozygous inactivation of the VHL gene in a G1 tumor support the notion that the inactivation of the VHL gene is an early event in tumorigenesis of renal cell carcinoma.