iNOS localizes to both the cytosol and peroxisomes in hepatocytes in vitro and in vivo. The structural determinants for iNOS localization are not known. One plausible mechanism for iNOS localization to the peroxisome is through the interaction with peroxisomal import proteins PEX5 or PEX7. siRNA knockdown of PEX7 reduced iNOS colocalization with the peroxisomal protein PMP70. Proteomic studies using MALDI-MS identified iNOS association with the 50-kD ezrin binding PDZ protein (EBP50). Confocal microscopy studies and immunoelectron microscopy confirmed iNOS association with EBP50, with greatest colocalization occurring at 8 hours of cytokine exposure. EBP50 associated with peroxisomes in a PEX5 and PEX7-dependent manner. iNOS localization to peroxisomes was contingent on EBP50 expression in LPS-treated mice. Thus, iNOS targeting to peroxisomes in hepatocytes involves interaction with PEX7 and EBP50. The targeting of iNOS protein to the peroxisome may shift the balance of metabolic processes that rely on heme proteins susceptible to modification by radical oxygen and nitrogen radicals.
Inflammation; Sepsis; Inducible Nitric Oxide Synthase; Peroxisome; Liver; Subcellular Localization
Whole-genome sequencing may revolutionize medical diagnostics through rapid identification of alleles that cause disease. However, even in cases with simple patterns of inheritance and unambiguous diagnoses, the relationship between disease phenotypes and their corresponding genetic changes can be complicated. Comprehensive diagnostic assays must therefore identify all possible DNA changes in each haplotype and determine which are responsible for the underlying disorder. The high number of rare, heterogeneous mutations present in all humans and the paucity of known functional variants in more than 90% of annotated genes make this challenge particularly difficult. Thus, the identification of the molecular basis of a genetic disease by means of whole-genome sequencing has remained elusive. We therefore aimed to assess the usefulness of human whole-genome sequencing for genetic diagnosis in a patient with Charcot–Marie–Tooth disease.
We identified a family with a recessive form of Charcot–Marie–Tooth disease for which the genetic basis had not been identified. We sequenced the whole genome of the proband, identified all potential functional variants in genes likely to be related to the disease, and genotyped these variants in the affected family members.
We identified and validated compound, heterozygous, causative alleles in SH3TC2 (the SH3 domain and tetratricopeptide repeats 2 gene), involving two mutations, in the proband and in family members affected by Charcot–Marie–Tooth disease. Separate subclinical phenotypes segregated independently with each of the two mutations; heterozygous mutations confer susceptibility to neuropathy, including the carpal tunnel syndrome.
As shown in this study of a family with Charcot–Marie–Tooth disease, whole-genome sequencing can identify clinically relevant variants and provide diagnostic information to inform the care of patients.
Algorithm-based exposure assessments based on patterns in questionnaire responses and professional judgment can readily apply transparent exposure decision rules to thousands of jobs quickly. However, we need to better understand how algorithms compare to a one-by-one job review by an exposure assessor. We compared algorithm-based estimates of diesel exhaust exposure to those of three independent raters within the New England Bladder Cancer Study, a population-based case–control study, and identified conditions under which disparities occurred in the assessments of the algorithm and the raters.
Occupational diesel exhaust exposure was assessed previously using an algorithm and a single rater for all 14 983 jobs reported by 2631 study participants during personal interviews conducted from 2001 to 2004. Two additional raters independently assessed a random subset of 324 jobs that were selected based on strata defined by the cross-tabulations of the algorithm and the first rater’s probability assessments for each job, oversampling their disagreements. The algorithm and each rater assessed the probability, intensity and frequency of occupational diesel exhaust exposure, as well as a confidence rating for each metric. Agreement among the raters, their aggregate rating (average of the three raters’ ratings) and the algorithm were evaluated using proportion of agreement, kappa and weighted kappa (κw). Agreement analyses on the subset used inverse probability weighting to extrapolate the subset to estimate agreement for all jobs. Classification and Regression Tree (CART) models were used to identify patterns in questionnaire responses that predicted disparities in exposure status (i.e., unexposed versus exposed) between the first rater and the algorithm-based estimates.
For the probability, intensity and frequency exposure metrics, moderate to moderately high agreement was observed among raters (κw = 0.50–0.76) and between the algorithm and the individual raters (κw = 0.58–0.81). For these metrics, the algorithm estimates had consistently higher agreement with the aggregate rating (κw = 0.82) than with the individual raters. For all metrics, the agreement between the algorithm and the aggregate ratings was highest for the unexposed category (90–93%) and was poor to moderate for the exposed categories (9–64%). Lower agreement was observed for jobs with a start year <1965 versus ≥1965. For the confidence metrics, the agreement was poor to moderate among raters (κw = 0.17–0.45) and between the algorithm and the individual raters (κw = 0.24–0.61). CART models identified patterns in the questionnaire responses that predicted a fair-to-moderate (33–89%) proportion of the disagreements between the raters’ and the algorithm estimates.
The agreement between any two raters was similar to the agreement between an algorithm-based approach and individual raters, providing additional support for using the more efficient and transparent algorithm-based approach. CART models identified some patterns in disagreements between the first rater and the algorithm. Given the absence of a gold standard for estimating exposure, these patterns can be reviewed by a team of exposure assessors to determine whether the algorithm should be revised for future studies.
case–control; diesel exhaust; expert judgement; exposure assessment
We conducted a pilot randomized clinical trial of office-based active vision therapy for the treatment of childhood amblyopia to determine the feasibility of conducting a full-scale randomized clinical trial.
A training and certification program and manual of procedures were developed to certify therapists to administer a standardized vision therapy program in ophthalmology and optometry offices consisting of weekly visits for 16 weeks. Nineteen children, 7 to less than 13 years of age, with amblyopia (20/40–20/100) were randomly assigned to receive either 2 hours of daily patching with active vision therapy or 2 hours of daily patching with placebo vision therapy.
Therapists in diverse practice settings were successfully trained and certified to perform standardized vision therapy in strict adherence with protocol. Subjects completed 85% of required weekly in-office vision therapy visits. Eligibility criteria based on age, visual acuity, and stereoacuity, designed to identify children able to complete a standardized vision therapy program and judged likely to benefit from this treatment, led to a high proportion of screened subjects being judged ineligible, resulting in insufficient recruitment. There were difficulties in retrieving adherence data for the computerized home therapy procedures.
This study demonstrated that a 16-week treatment trial of vision therapy was feasible with respect to maintaining protocol adherence; however, recruitment under the proposed eligibility criteria, necessitated by the standardized approach to vision therapy, was not successful. A randomized clinical trial of in-office vision therapy for the treatment of amblyopia would require broadening of the eligibility criteria and improved methods to gather objective data regarding the home therapy. A more flexible approach that customizes vision therapy based on subject age, visual acuity, and stereopsis, might be required to allow enrollment of a broader group of subjects.
amblyopia; patching; vision therapy; placebo vision therapy; masking
Evaluating occupational exposures in population-based case-control studies often requires exposure assessors to review each study participants' reported occupational information job-by-job to derive exposure estimates. Although such assessments likely have underlying decision rules, they usually lack transparency, are time-consuming and have uncertain reliability and validity. We aimed to identify the underlying rules to enable documentation, review, and future use of these expert-based exposure decisions.
Classification and regression trees (CART, predictions from a single tree) and random forests (predictions from many trees) were used to identify the underlying rules from the questionnaire responses and an expert's exposure assignments for occupational diesel exhaust exposure for several metrics: binary exposure probability and ordinal exposure probability, intensity, and frequency. Data were split into training (n=10,488 jobs), testing (n=2,247), and validation (n=2,248) data sets.
The CART and random forest models' predictions agreed with 92–94% of the expert's binary probability assignments. For ordinal probability, intensity, and frequency metrics, the two models extracted decision rules more successfully for unexposed and highly exposed jobs (86–90% and 57–85%, respectively) than for low or medium exposed jobs (7–71%).
CART and random forest models extracted decision rules and accurately predicted an expert's exposure decisions for the majority of jobs and identified questionnaire response patterns that would require further expert review if the rules were applied to other jobs in the same or different study. This approach makes the exposure assessment process in case-control studies more transparent and creates a mechanism to efficiently replicate exposure decisions in future studies.
diesel exhaust; classification; data mining; occupational exposure
Although acute lymphocytic leukemia (ALL) is the most common childhood cancer, genetic predisposition to ALL remains poorly understood. Whole-exome sequencing was performed in an extended kindred in which five individuals had been diagnosed with leukemia. Analysis revealed a nonsense variant of TP53 which has been previously reported in families with sarcomas and other typical Li Fraumeni syndrome-associated cancers but never in a familial leukemia kindred. This unexpected finding enabled identification of an appropriate sibling bone marrow donor and illustrates that exome sequencing will reveal atypical clinical presentations of even well-studied genes.
exome sequencing; acute lymphocytic leukemia; genetic predisposition to disease; genetic testing
Approximately 15% of colorectal carcinomas (CRC) exhibit a hypermutated genotype accompanied by high levels of microsatellite instability (MSI-H) and defects in DNA mismatch repair. These tumors, unlike the majority of colorectal carcinomas, are often diploid, exhibit frequent epigenetic silencing of the MLH1 DNA mismatch repair gene, and have a better clinical prognosis. As an adjunct study to The Cancer Genome Atlas consortium that recently analyzed 224 colorectal cancers by whole exome sequencing, we compared the 35 CRC (15.6%) with a hypermutated genotype to those with a non-hypermutated genotype. We found that 22 (63%) of hypermutated CRC exhibited transcriptional silencing of the MLH1 gene, a high frequency of BRAF V600E gene mutations and infrequent APC and KRAS mutations, a mutational pattern significantly different from their non-hypermutated counterparts. However, the remaining 13 (37%) hypermutated CRC lacked MLH1 silencing, contained tumors with the highest mutation rates (“ultramutated” CRC), and exhibited higher incidences of APC and KRAS mutations, but infrequent BRAF mutations. These patterns were confirmed in an independent validation set of 250 exome-sequenced CRC. Analysis of mRNA and microRNA expression signatures revealed that hypermutated CRC with MLH1 silencing had greatly reduced levels of WNT signaling and increased BRAF signaling relative non-hypermutated CRC. Our findings suggest that hypermutated CRC include one subgroup with fundamentally different pathways to malignancy than the majority of CRC. Examination of MLH1 expression status and frequencies of APC, KRAS, and BRAF mutation in CRC may provide a useful diagnostic tool that could supplement the standard microsatellite instability assays and influence therapeutic decisions.
colorectal cancer; microsatellite instability; MLH1; APC; KRAS; BRAF; WNT signaling; mutation rate
Genetic mapping on fully sequenced individuals is transforming our understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating novel genes in models of anxiety, heart disease and multiple sclerosis. The relation between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show the extent and spatial pattern of variation in inbred rats differ significantly from those of inbred mice, and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.
Fig pollinating wasps form obligate symbioses with their fig hosts. This mutualism arose approximately 75 million years ago. Unlike many other intimate symbioses, which involve vertical transmission of symbionts to host offspring, female fig wasps fly great distances to transfer horizontally between hosts. In contrast, male wasps are wingless and cannot disperse. Symbionts that keep intimate contact with their hosts often show genome reduction, but it is not clear if the wide dispersal of female fig wasps will counteract this general tendency. We sequenced the genome of the fig wasp Ceratosolen solmsi to address this question.
The genome size of the fig wasp C. solmsi is typical of insects, but has undergone dramatic reductions of gene families involved in environmental sensing and detoxification. The streamlined chemosensory ability reflects the overwhelming importance of females finding trees of their only host species, Ficus hispida, during their fleeting adult lives. Despite long-distance dispersal, little need exists for detoxification or environmental protection because fig wasps spend nearly all of their lives inside a largely benign host. Analyses of transcriptomes in females and males at four key life stages reveal that the extreme anatomical sexual dimorphism of fig wasps may result from a strong bias in sex-differential gene expression.
Our comparison of the C. solmsi genome with other insects provides new insights into the evolution of obligate mutualism. The draft genome of the fig wasp, and transcriptomic comparisons between both sexes at four different life stages, provide insights into the molecular basis for the extreme anatomical sexual dimorphism of this species.
Sensitive indicators of spatial and temporal variation in vector-host contact rates are critical to understanding the transmission and eventual prevention of arboviruses such as West Nile virus (WNV). Monitoring vector contact rates on particularly susceptible and perhaps more exposed avian nestlings may provide an advanced indication of local WNV amplification. To test this hypothesis we monitored WNV infection and vector contact rates among nestlings occupying nest boxes (primarily Eastern bluebirds; Sialia sialis, Turdidae) across Henrico County, Virginia, USA, from May to August 2012. Observed host-seeking rates were temporally variable and associated with absolute vector and host abundances. Despite substantial effort to monitor WNV among nestlings and mosquitoes, we did not detect the presence of WNV in these populations. Generally low vector-nestling host contact rates combined with the negative WNV infection data suggest that monitoring transmission parameters among nestling Eastern bluebirds in Henrico County, Virginia, USA may not be a sensitive indicator of WNV activity.
host-seeking rate; nestling; nest mosquito trap; arbovirus; West Nile virus
The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5′ regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5′ and 3′ UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression.
Insects use methylation to modulate genome function in a different manner from vertebrates. Here, we quantified the global methylation profile in a parasitic wasp species, Nasonia vitripennis, a model with some advantages over ant and honeybee for functional and genetic analyses of methylation, such as short generation time, inbred lines, and inter-fertile species. Using a highly inbred line permitted us to precisely characterize DNA methylation, which is compared to gene expression variation across developmental stages, and contrasted to other insect species. DNA methylation is almost exclusively on the 5′-most 1 kbp coding exons, and ∼1/3 of protein coding genes are methylated. Methylated genes tend to occur in small clusters in the genome. Unlike many organisms, Nasonia leaves nearly all transposable element genes non-methylated. Methylated genes exhibit more uniform expression across developmental stages for both moderately and highly expressed genes, suggesting that DNA methylation is marking the genes for constitutive expression. Among pairs of differentially methylated duplicated genes, the paralogs that lose DNA methylation after duplication in the Nasonia lineage show lower expression and greater specialization of expression. Finally, by comparative analysis, we show that methylated genes are more conserved at three different time scales during evolution.
Professional judgment is necessary to assess occupational exposure in population-based case-control studies; however, the assessments lack transparency and are time-consuming to perform. To improve transparency and efficiency, we systematically applied decision rules to the questionnaire responses to assess diesel exhaust exposure in the New England Bladder Cancer Study, a population-based case-control study.
2,631 participants reported 14,983 jobs; 2,749 jobs were administered questionnaires (‘modules’) with diesel-relevant questions. We applied decision rules to assign exposure metrics based solely on the occupational history responses (OH estimates) and based on the module responses (module estimates); we combined the separate OH and module estimates (OH/module estimates). Each job was also reviewed one at a time to assign exposure (one-by-one review estimates). We evaluated the agreement between the OH, OH/module, and one-by-one review estimates.
The proportion of exposed jobs was 20–25% for all jobs, depending on approach, and 54–60% for jobs with diesel-relevant modules. The OH/module and one-by-one review had moderately high agreement for all jobs (κw=0.68–0.81) and for jobs with diesel-relevant modules (κw=0.62–0.78) for the probability, intensity, and frequency metrics. For exposed subjects, the Spearman correlation statistic was 0.72 between the cumulative OH/module and one-by-one review estimates.
The agreement seen here may represent an upper level of agreement because the algorithm and one-by-one review estimates were not fully independent. This study shows that applying decision-based rules can reproduce a one-by-one review, increase transparency and efficiency, and provide a mechanism to replicate exposure decisions in other studies.
Ecologic studies have reported that solar ultraviolet radiation (UVR) exposure is associated with cancer, but little evidence is available from prospective studies. We aimed to assess the association between an objective measure of ambient UVR exposure and risk of total and site-specific cancer in a large, regionally diverse cohort (450,934 white, non-Hispanic subjects (50-71 years old) in the prospective NIH-AARP Diet and Health Study) after accounting for individual-level confounding risk factors. Estimated erythemal UVR exposure from satellite Total Ozone Mapping Spectrometer (TOMS) data from NASA was linked to the U.S. Census Bureau 2000 census tract (centroid) of baseline residence for each subject. We used Cox proportional hazards models adjusted for multiple potential confounders to estimate hazard ratios (HR) and 95% confidence intervals (CI) for quartiles of UVR exposure. Restricted cubic splines examined non-linear relationships. Over 9 years of follow-up, UVR exposure was inversely associated with total cancer risk (N=75,917; highest vs. lowest quartile, HR=0.97 (0.95, 0.99), p-trend<0.001). In site-specific cancer analyses, UVR exposure was associated with increased melanoma risk (highest vs. lowest quartile, HR=1.22 (1.13, 1.32), p-trend<0.001) and decreased risk of Non-Hodgkin’s lymphoma (HR=0.82 (0.74, 0.92)) and colon (HR=0.88 (0.82, 0.96)), squamous cell lung (HR=0.86 (0.75, 0.98)), pleural (HR=0.57 (0.38, 0.84)), prostate (HR=0.91 (0.88, 0.95)), kidney (HR=0.83 (0.73, 0.94)), and bladder (HR=0.88 (0.81, 0.96)) cancers (all p-trend<0.05). We also found non-linear associations for some cancer sites, including the thyroid and pancreas. Our results add to mounting evidence for the influential role of UVR exposure on cancer.
Ultraviolet radiation; cancer; vitamin D; prospective
Exploring spatial-temporal patterns of disease incidence and mortality can identify areas of significantly elevated or decreased risk, providing potential etiologic clues. Several methodological issues arise in spatial-temporal analysis of cancer, including population mobility, disease latency, and confounding, but applying modern statistical methods to case-control studies with residential histories can address these issues. As an example, we present a spatial-temporal analysis of non-Hodgkin lymphoma (NHL) risk using data from Los Angeles County, one of four centers in a population-based case-control study. Using residential histories, we fitted generalized additive models (GAMs) adjusted for known risk factors to model spatially the probability that an individual had NHL and identify areas of significantly elevated NHL risk. In previous analyses using models with single lag times, the lag time of 20 years yielded the most significant decrease in model deviance. To better assess cumulative effects of unmeasured environmental exposures over space and time, we considered models that allowed for multiple residences per subject through spatial smoothing functions of residential location at different times. We found that the model with the best goodness-of-fit included components for residential change and residential duration, although the model that included residential duration was not meaningfully better than the model that included only residential change. The estimated cumulative spatial risk surface from the model with residential change amplified the risk surface in some areas compared with the surface based on the model with a single component for the most significant time lag.
cancer; generalized additive model; spatial risk; latency; exposure
Transposable elements (TEs) are abundant in the human genome, and some are capable of generating new insertions through RNA intermediates. In cancer, the disruption of cellular mechanisms that normally suppress TE activity may facilitate mutagenic retrotranspositions. We performed single-nucleotide resolution analysis of TE insertions in 43 high-coverage whole-genome sequencing data sets from five cancer types. We identified 194 high-confidence somatic TE insertions, as well as thousands of polymorphic TE insertions in matched normal genomes. Somatic insertions were present in epithelial tumors but not in blood or brain cancers. Somatic L1 insertions tend to occur in genes that are commonly mutated in cancer, disrupt the expression of the target genes, and are biased toward regions of cancer-specific DNA hypomethylation, highlighting their potential impact in tumorigenesis.
Bacteria to eukaryote lateral gene transfers (LGT) are an important potential source of material for the evolution of novel genetic traits. The explosion in the number of newly sequenced genomes provides opportunities to identify and characterize examples of these lateral gene transfer events, and to assess their role in the evolution of new genes. In this paper, we describe an ancient lepidopteran LGT of a glycosyl hydrolase family 31 gene (GH31) from an Enterococcus bacteria. PCR amplification between the LGT and a flanking insect gene confirmed that the GH31 was integrated into the Bombyx mori genome and was not a result of an assembly error. Database searches in combination with degenerate PCR on a panel of 7 lepidopteran families confirmed that the GH31 LGT event occurred deep within the Order approximately 65–145 million years ago. The most basal species in which the LGT was found is Plutella xylostella (superfamily: Yponomeutoidea). Array data from Bombyx mori shows that GH31 is expressed, and low dN/dS ratios indicates the LGT coding sequence is under strong stabilizing selection. These findings provide further support for the proposition that bacterial LGTs are relatively common in insects and likely to be an underappreciated source of adaptive genetic material.
schizophrenia; sequencing; SNV; genetic; association; mutation; DISC1
Individual exposure to ultraviolet radiation (UVR) is challenging to measure, particularly for diseases with substantial latency periods between first exposure and diagnosis of outcome, such as cancer. To guide the choice of surrogates for long-term UVR exposure in epidemiologic studies, we assessed how well stable sun-related individual characteristics and environmental/meteorological factors predicted daily personal UVR exposure measurements.
We evaluated 123 United States Radiologic Technologists subjects who wore personal UVR dosimeters for 8 hours daily for up to 7 days (N = 837 days). Potential predictors of personal UVR derived from a self-administered questionnaire, and public databases that provided daily estimates of ambient UVR and weather conditions. Factors potentially related to personal UVR exposure were tested individually and in a model including all significant variables.
The strongest predictors of daily personal UVR exposure in the full model were ambient UVR, latitude, daily rainfall, and skin reaction to prolonged sunlight (R2 = 0.30). In a model containing only environmental and meteorological variables, ambient UVR, latitude, and daily rainfall were the strongest predictors of daily personal UVR exposure (R2 = 0.25).
In the absence of feasible measures of individual longitudinal sun exposure history, stable personal characteristics, ambient UVR, and weather parameters may help estimate long-term personal UVR exposure.
The development of a microneedle-based biosensor array for multiplexed in situ detection of exercise-induced metabolic acidosis, tumor microenvironment, and other variations in tissue chemistry is described. Simultaneous and selective amperometric detection of pH, glucose, and lactate over a range of physiologically-relevant concentrations in complex media is demonstrated. Furthermore, materials modified with a cell-resistant (Lipidure®) coating were shown to inhibit macrophage adhesion; no signs of coating delamination were noted over a 48-hour period.
microneedle biosensor; microneedle; multiplexed detection; tumor microenvironment; carbon paste
Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.
To characterize tumor growth and metastatic potential in head and neck squamous cell carcinoma (HNSCC) cell lines in an orthotopic murine model of oral tongue cancer, and to correlate TP53 mutation status with these findings.
Cells from each of 48 HNSCC cell lines were orthotopically injected into the oral tongues of nude mice. Tumor volume, cervical lymph node metastasis, and mouse survival were recorded. Direct sequencing of the TP53 gene and western blot analysis for the p53 protein after induction with 5-fluorouracil was performed. Cell lines were categorized as either mutant TP53 or wild-type TP53, and lines with TP53 mutation were further categorized on the basis of type of mutation (disruptive or non-disruptive), and level of p53 protein expression. The behavior of tumors in these different groups was compared.
The 48 HNSCC cell lines showed a wide range of behavior from highly aggressive and metastatic to no tumor formation. Mice injected with cells harboring disruptive TP53 mutations had faster tumor growth, greater incidence of cervical lymph node metastasis, and shorter survival than mice injected with cells lacking these mutations.
HNSCC cell lines display a wide spectrum of behavior in an orthotopic model of oral cancer. Cell lines with disruptive TP53 mutations are more aggressive in this system, corroborating clinical reports that have linked these mutations to poor patient outcome.
head and neck squamous cell carcinoma; TP53; disruptive TP53 mutation; cervical lymph node metastasis; orthotopic nude mouse model
Ion channel mutations are an important cause of rare Mendelian disorders affecting brain, heart, and other tissues. We performed parallel exome sequencing of 237 channel genes in a well characterized human sample, comparing variant profiles of unaffected individuals to those with the most common neuronal excitability disorder, sporadic idiopathic epilepsy. Rare missense variation in known Mendelian disease genes is prevalent in both groups at similar complexity, revealing that even deleterious ion channel mutations confer uncertain risk to an individual depending on the other variants with which they are combined. Our findings indicate that variant discovery via large scale sequencing efforts is only a first step in illuminating the complex allelic architecture underlying personal disease risk. We propose that in silico modeling of channel variation in realistic cell and network models will be crucial to future strategies assessing mutation profile pathogenicity and drug response in individuals with a broad spectrum of excitability disorders.
The Cancer Genome Atlas (TCGA) Network recently comprehensively catalogued the molecular aberrations in 487 high-grade serous ovarian cancers, with much remaining to be elucidated regarding the microRNAs (miRNAs). Here, using TCGA ovarian data, we surveyed the miRNAs, in the context of their predicted gene targets.
Methods and Results
Integration of miRNA and gene patterns yielded evidence that proximal pairs of miRNAs are processed from polycistronic primary transcripts, and that intronic miRNAs and their host gene mRNAs derive from common transcripts. Patterns of miRNA expression revealed multiple tumor subtypes and a set of 34 miRNAs predictive of overall patient survival. In a global analysis, miRNA:mRNA pairs anti-correlated in expression across tumors showed a higher frequency of in silico predicted target sites in the mRNA 3′-untranslated region (with less frequency observed for coding sequence and 5′-untranslated regions). The miR-29 family and predicted target genes were among the most strongly anti-correlated miRNA:mRNA pairs; over-expression of miR-29a in vitro repressed several anti-correlated genes (including DNMT3A and DNMT3B) and substantially decreased ovarian cancer cell viability.
This study establishes miRNAs as having a widespread impact on gene expression programs in ovarian cancer, further strengthening our understanding of miRNA biology as it applies to human cancer. As with gene transcripts, miRNAs exhibit high diversity reflecting the genomic heterogeneity within a clinically homogeneous disease population. Putative miRNA:mRNA interactions, as identified using integrative analysis, can be validated. TCGA data are a valuable resource for the identification of novel tumor suppressive miRNAs in ovarian as well as other cancers.