Epidemiology of Doublet/Multiplet Mutations in Lung Cancers: Evidence that a Subset Arises by Chronocoordinate Events

BACKGROUND: Evidence strongly suggests that spontaneous doublet mutations in normal mouse tissues generally arise from chronocoordinate events. These chronocoordinate mutations sometimes reflect "mutation showers", which are multiple chronocoordinate mutations spanning many kilobases. However, little is known about mutagenesis of doublet and multiplet mutations (domuplets) in human cancer. Lung cancer accounts for about 25% of all cancer deaths. Herein, we analyze the epidemiology of domuplets in the EGFR and TP53 genes in lung cancer. The EGFR gene is an oncogene in which doublets are generally driver plus driver mutations, while the TP53 gene is a tumor suppressor gene with a more typical situation in which doublets derive from a driver and passenger mutation. METHODOLOGY/PRINCIPAL FINDINGS: EGFR mutations identified by sequencing were collected from 66 published papers and our updated EGFR mutation database (www.egfr.org). TP53 mutations were collected from IARC version 12 (www-p53.iarc.fr). For EGFR and TP53 doublets, no clearly significant differences in race, ethnicity, gender and smoking status were observed. Doublets in the EGFR and TP53 genes in human lung cancer are elevated about eight- and three-fold, respectively, relative to spontaneous doublets in mouse (6% and 2.3% versus 0.7%). CONCLUSIONS/SIGNIFICANCE: Although no one characteristic is definitive, the aggregate properties of doublet and multiplet mutations in lung cancer are consistent with a subset derived from chronocoordinate events in the EGFR gene: i) the eight frameshift doublets (present in 0.5% of all patients with EGFR mutations) are clustered and produce a net in-frame change; ii) about 32% of doublets are very closely spaced (< or =30 nt); and iii) multiplets contain two or more closely spaced mutations. TP53 mutations in lung cancer are very closely spaced (< or =30 nt) in 33% of doublets, and multiplets generally contain two or more very closely spaced mutations. Work in model systems is necessary to confirm the significance of chronocoordinate events in lung and other cancers

Trim17, novel E3 ubiquitin-ligase, initiates neuronal apoptosis

Accumulating data indicate that the ubiquitin-proteasome system controls apoptosis by regulating the level and the function of key regulatory proteins. In this study, we identified Trim17, a member of the TRIM/RBCC protein family, as one of the critical E3 ubiquitin ligases involved in the control of neuronal apoptosis upstream of mitochondria. We show that expression of Trim17 is increased both at the mRNA and protein level in several in vitro models of transcription-dependent neuronal apoptosis. Expression of Trim17 is controlled by the PI3K/Akt/GSK3 pathway in cerebellar granule neurons (CGN). Moreover, the Trim17 protein is expressed in vivo, in apoptotic neurons that naturally die during post-natal cerebellar development. Overexpression of active Trim17 in primary CGN was sufficient to induce the intrinsic pathway of apoptosis in survival conditions. This pro-apoptotic effect was abolished in Bax(-/-) neurons and depended on the E3 activity of Trim17 conferred by its RING domain. Furthermore, knock-down of endogenous Trim17 and overexpression of dominant-negative mutants of Trim17 blocked trophic factor withdrawal-induced apoptosis both in CGN and in sympathetic neurons. Collectively, our data are the first to assign a cellular function to Trim17 by showing that its E3 activity is both necessary and sufficient for the initiation of neuronal apoptosis. Cell Death and Differentiation (2010) 17, 1928-1941; doi: 10.1038/cdd.2010.73; published online 18 June 201

Kita Driven Expression of Oncogenic HRAS Leads to Early Onset and Highly Penetrant Melanoma in Zebrafish

Melanoma is the most aggressive and lethal form of skin cancer. Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed.Using the combinatorial Gal4-UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter. Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth. By 2-4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish. The adult tumors evident between 1-3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line. Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors. In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period.This indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors. Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens

A statistical method for excluding non-variable CpG sites in high-throughput DNA methylation profiling

<p>Abstract</p> <p>Background</p> <p>High-throughput DNA methylation arrays are likely to accelerate the pace of methylation biomarker discovery for a wide variety of diseases. A potential problem with a standard set of probes measuring the methylation status of CpG sites across the whole genome is that many sites may not show inter-individual methylation variation among the biosamples for the disease outcome being studied. Inclusion of these so-called "non-variable sites" will increase the risk of false discoveries and reduce statistical power to detect biologically relevant methylation markers.</p> <p>Results</p> <p>We propose a method to estimate the proportion of non-variable CpG sites and eliminate those sites from further analyses. Our method is illustrated using data obtained by hybridizing DNA extracted from the peripheral blood mononuclear cells of 311 samples to an array assaying 1505 CpG sites. Results showed that a large proportion of the CpG sites did not show inter-individual variation in methylation.</p> <p>Conclusions</p> <p>Our method resulted in a substantial improvement in association signals between methylation sites and outcome variables while controlling the false discovery rate at the same level.</p

Complications related to deep venous thrombosis prophylaxis in trauma: a systematic review of the literature

Deep venous thrombosis prophylaxis is essential to the appropriate management of multisystem trauma patients. Without thromboprophylaxis, the rate of venous thrombosis and subsequent pulmonary embolism is substantial. Three prophylactic modalities are common: pharmacologic anticoagulation, mechanical compression devices, and inferior vena cava filtration. A systematic review was completed using PRISMA guidelines to evaluate the potential complications of DVT prophylactic options. Level one evidence currently supports the use of low molecular weight heparins for thromboprophylaxis in the trauma patient. Unfortunately, multiple techniques are not infrequently required for complex multisystem trauma patients. Each modality has potential complications. The risks of heparin include bleeding and heparin induced thrombocytopenia. Mechanical compression devices can result in local soft tissue injury, bleeding and patient non-compliance. Inferior vena cava filters migrate, cause inferior vena cava occlusion, and penetrate the vessel wall. While the use of these techniques can be life saving, they must be appropriately utilized

Mutations of the BRCA1 and BRCA2 genes in patients with bilateral breast cancer

Mutations of the BRCA1 or BRCA2 genes have been shown to strongly predispose towards the development of contralateral breast cancer in patients from large multi-case families. In order to test the hypothesis that BRCA1 and BRCA2 mutations are more frequent in patients with bilateral breast cancer, we have investigated a hospital-based series of 75 consecutive patients with bilateral breast cancer and a comparison group of 75 patients with unilateral breast cancer, pairwise matched by age and family history, for mutations in the BRCA1 and BRCA2 genes. Five frameshift deletions (517delGT in BRCA1; 4772delA, 5946delCT, 6174delT and 8138del5 in BRCA2) were identified in patients with bilateral disease. No further mutations, apart from polymorphisms and 3 rare unclassified variants, were found after scanning the whole BRCA1 and BRCA2 coding sequence. Three pathogenic BRCA1 mutations (Cys61Gly, 3814del5, 5382insC) were identified in the group of patients with unilateral breast cancer. The frequencies of common BRCA1 and BRCA2 missense variants were not different between the 2 groups. In summary, we did not find a significantly increased prevalence of BRCA1 and BRCA2 mutations in a hospital-based cohort of German patients with bilateral breast cancer. We conclude that bilaterality of breast cancer on its own is not strongly associated with BRCA1 and BRCA2 mutations when adjusted for age and family history. The high frequency of bilateral disease in multi-case breast cancer families may be due to a familial aggregation of additional susceptibility factors modifying the penetrance of BRCA1 and BRCA2 mutations. © 2001 Cancer Research Campaignhttp://www.bjcancer.co

Improving Salmonella vector with rec mutation to stabilize the DNA cargoes

<p>Abstract</p> <p>Background</p> <p><it>Salmonella </it>has been employed to deliver therapeutic molecules against cancer and infectious diseases. As the carrier for target gene(s), the cargo plasmid should be stable in the bacterial vector. Plasmid recombination has been reduced in <it>E. coli </it>by mutating several genes including the <it>recA</it>, <it>recE</it>, <it>recF </it>and <it>recJ</it>. However, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in <it>Salmonella enterica</it>.</p> <p>Results</p> <p>The effect of <it>recA</it>, <it>recF </it>and <it>recJ </it>deletions on DNA recombination was examined in three serotypes of <it>Salmonella enterica</it>. We found that (1) intraplasmid recombination between direct duplications was RecF-independent in Typhimurium and Paratyphi A, but could be significantly reduced in Typhi by a Δ<it>recA </it>or Δ<it>recF </it>mutation; (2) in all three <it>Salmonella </it>serotypes, both Δ<it>recA </it>and Δ<it>recF </it>mutations reduced intraplasmid recombination when a 1041 bp intervening sequence was present between the duplications; (3) Δ<it>recA </it>and Δ<it>recF </it>mutations resulted in lower frequencies of interplasmid recombination in Typhimurium and Paratyphi A, but not in Typhi; (4) in some cases, a Δ<it>recJ </it>mutation could reduce plasmid recombination but was less effective than Δ<it>recA </it>and Δ<it>recF </it>mutations. We also examined chromosome-related recombination. The frequencies of intrachromosomal recombination and plasmid integration into the chromosome were 2 and 3 logs lower than plasmid recombination frequencies in Rec⁺strains. A Δ<it>recA </it>mutation reduced both intrachromosomal recombination and plasmid integration frequencies.</p> <p>Conclusions</p> <p>The Δ<it>recA </it>and Δ<it>recF </it>mutations can reduce plasmid recombination frequencies in <it>Salmonella enterica</it>, but the effect can vary between serovars. This information will be useful for developing <it>Salmonella </it>delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy.</p

Evaluating the Number of Stages in Development of Squamous Cell and Adenocarcinomas across Cancer Sites Using Human Population-Based Cancer Modeling

BACKGROUND: Adenocarcinomas (ACs) and squamous cell carcinomas (SCCs) differ by clinical and molecular characteristics. We evaluated the characteristics of carcinogenesis by modeling the age patterns of incidence rates of ACs and SCCs of various organs to test whether these characteristics differed between cancer subtypes. METHODOLOGY/PRINCIPAL FINDINGS: Histotype-specific incidence rates of 14 ACs and 12 SCCs from the SEER Registry (1973-2003) were analyzed by fitting several biologically motivated models to observed age patterns. A frailty model with the Weibull baseline was applied to each age pattern to provide the best fit for the majority of cancers. For each cancer, model parameters describing the underlying mechanisms of carcinogenesis including the number of stages occurring during an individual's life and leading to cancer (m-stages) were estimated. For sensitivity analysis, the age-period-cohort model was incorporated into the carcinogenesis model to test the stability of the estimates. For the majority of studied cancers, the numbers of m-stages were similar within each group (i.e., AC and SCC). When cancers of the same organs were compared (i.e., lung, esophagus, and cervix uteri), the number of m-stages were more strongly associated with the AC/SCC subtype than with the organ: 9.79±0.09, 9.93±0.19 and 8.80±0.10 for lung, esophagus, and cervical ACs, compared to 11.41±0.10, 12.86±0.34 and 12.01±0.51 for SCCs of the respective organs (p<0.05 between subtypes). Most SCCs had more than ten m-stages while ACs had fewer than ten m-stages. The sensitivity analyses of the model parameters demonstrated the stability of the obtained estimates. CONCLUSIONS/SIGNIFICANCE: A model containing parameters capable of representing the number of stages of cancer development occurring during individual's life was applied to the large population data on incidence of ACs and SCCs. The model revealed that the number of m-stages differed by cancer subtype being more strongly associated with ACs/SCCs histotype than with organ/site