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1.  ARTD1/PARP1 negatively regulates glycolysis by inhibiting hexokinase 1 independent of NAD+ depletion 
Cell reports  2014;8(6):1819-1831.
Summary
ARTD1 (PARP1) is a key enzyme involved in DNA repair by synthesizing poly(ADP-ribose) (PAR) in response to strand breaks and plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD+ depletion and ATP loss, however the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we directly compared the effects of ARTD1 activation and direct NAD+ depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD+ depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics based PAR-interactome after DNA damage and identified hexokinase 1 (HK1) as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing new insight on the importance of nucleus to mitochondria communication via ARTD1 activation.
doi:10.1016/j.celrep.2014.08.036
PMCID: PMC4177344  PMID: 25220464
PARP; DNA damage; metabolism; glycolysis; Hexokinase 1; NAD+; ATP; ARTD1
3.  Environmental determinants of polychlorinated biphenyl concentrations in residential carpet dust 
Environmental science & technology  2013;47(18):10405-10414.
Polychlorinated biphenyls (PCBs), banned in the United Sates in the late 1970s, are still found in indoor and outdoor environments. Little is known about the determinants of PCB levels in homes. We measured concentrations of 5 PCB congeners (105, 138, 153, 170, 180) in carpet dust collected between 1998–2000 from 1,187 homes in four sites: Detroit, Iowa, Los Angeles, and Seattle. Home characteristics, occupational history, and demographic information were obtained by interview. We used a geographic information system to geocode addresses and determine distances to the nearest major road, freight route, and railroad, percentage of developed land, number of industrial facilities within 2 km of residences, and population density. Ordinal logistic regression was used to estimate the associations between the covariates of interest and the odds of PCB detection in each site separately. Total PCBs levels (all congeners < maximum practical quantitation limit [MPQL] vs. at least one congener ≥ MPQL to < median concentration vs. at least one congener >median concentration) were positively associated with either percentage of developed land (ORrange: 1.01-1.04 for each percentage increase) or population density (OR: 1.08 for every 1,000/mi2) in each site. The number of industrial facilities within 2 km of a home was associated with PCB concentrations; however, facility type and the direction of the association varied by site. Our findings suggest that outdoor sources of PCBs may be significant determinants of indoor concentrations.
doi:10.1021/es401447w
PMCID: PMC4076890  PMID: 23952055
4.  Amphetamine Modulates Glutamatergic Neurotransmission through Endocytosis of the Excitatory Amino Acid Transporter EAAT3 in Dopamine Neurons 
Neuron  2014;83(2):404-416.
Summary
Amphetamines modify the brain and alter behavior through mechanisms generally attributed to their ability to regulate extracellular dopamine concentrations. However, the actions of amphetamine are also linked to adaptations in glutamatergic signaling. We report here that when amphetamine enters dopamine neurons through the dopamine transporter, it stimulates endocytosis of an excitatory amino acid transporter, EAAT3, in dopamine neurons in vitro and in vivo. Consistent with this decrease in surface EAAT3, amphetamine potentiates excitatory synaptic responses in dopamine neurons. We also show that the process of internalization is dynamin- and Rho-mediated and requires a unique sequence in the cytosolic C-terminus of EAAT3. Introduction of a peptide based on this motif into dopamine neurons blocks the effects of amphetamine on EAAT3 internalization and its action on excitatory responses. These data indicate that the internalization of EAAT3 triggered by amphetamine increases glutamatergic signaling and thus contributes to the effects of amphetamine on neurotransmission.
doi:10.1016/j.neuron.2014.05.043
PMCID: PMC4159050  PMID: 25033183
5.  Prediction of Missense Mutation Functionality Depends on both the Algorithm and Sequence Alignment Employed 
Human mutation  2011;32(6):661-668.
Multiple algorithms are used to predict the impact of missense mutations on protein structure and function using algorithm-generated sequence alignments or manually curated alignments. We compared the accuracy with native alignment of SIFT, Align-GVGD, PolyPhen-2 and Xvar when generating functionality predictions of well characterized missense mutations (n = 267) within the BRCA1, MSH2, MLH1 and TP53 genes. We also evaluated the impact of the alignment employed on predictions from these algorithms (except Xvar) when supplied the same four alignments including alignments automatically generated by (1) SIFT, (2) Polyphen-2, (3) Uniprot, and (4) a manually curated alignment tuned for Align-GVGD. Alignments differ in sequence composition and evolutionary depth. Data-based receiver operating characteristic curves employing the native alignment for each algorithm result in area under the curve of 78-79% for all four algorithms. Predictions from the PolyPhen-2 algorithm were least dependent on the alignment employed. In contrast, Align-GVGD predicts all variants neutral when provided alignments with a large number of sequences. Of note, algorithms make different predictions of variants even when provided the same alignment and do not necessarily perform best using their own alignment. Thus, researchers should consider optimizing both the algorithm and sequence alignment employed in missense prediction.
doi:10.1002/humu.21490
PMCID: PMC4154965  PMID: 21480434
multiple sequence alignment; SIFT; PolyPhen-2; Align-GVGD; Xvar; BRCA1; MSH2; MLH1; TP53
6.  SubcloneSeeker: a computational framework for reconstructing tumor clone structure for cancer variant interpretation and prioritization 
Genome Biology  2014;15(8):443.
Many tumors are composed of genetically divergent cell subpopulations. We report SubcloneSeeker, a package capable of exhaustive identification of subclone structures and evolutionary histories with bulk somatic variant allele frequency measurements from tumor biopsies. We present a statistical framework to elucidate whether specific sets of mutations are present within the same subclones, and the order in which they occur. We demonstrate how subclone reconstruction provides crucial information about tumorigenesis and relapse mechanisms; guides functional study by variant prioritization, and has the potential as a rational basis for informed therapeutic strategies for the patient. SubcloneSeeker is available at: https://github.com/yiq/SubcloneSeeker.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0443-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0443-x
PMCID: PMC4180956  PMID: 25160522
7.  Prospective Study of Ultraviolet Radiation Exposure and Mortality Risk in the United States 
American Journal of Epidemiology  2013;178(4):521-533.
Geographic variations in mortality rate in the United States could be due to several hypothesized factors, one of which is exposure to solar ultraviolet radiation (UVR). Limited evidence from previous prospective studies has been inconclusive. The association between ambient residential UVR exposure and total and cause-specific mortality risks in a regionally diverse cohort (346,615 white, non-Hispanic subjects, 50–71 years of age, in the National Institutes of Health (NIH)–AARP Diet and Health Study) was assessed, with accounting for individual-level confounders. UVR exposure (averaged for 1978–1993 and 1996–2005) from NASA's Total Ozone Mapping Spectrometer was linked to the US Census Bureau 2000 census tract of participants' baseline residence. Multivariate-adjusted Cox proportional-hazards models were used to estimate hazard ratios and 95% confidence intervals. Over 12 years, UVR exposure was associated with total deaths (n = 41,425; hazard ratio for highest vs. lowest quartiles (HRQ4 vs. Q1) = 1.06, 95% confidence interval (CI): 1.03, 1.09; Ptrend < 0.001) and with deaths (all Ptrend < 0.05) due to cancer (HRQ4 vs. Q1 = 1.06, 95% CI: 1.02, 1.11), cardiovascular disease (HRQ4 vs. Q1 = 1.06, 95% CI: 1.00, 1.12), respiratory disease (HRQ4 vs. Q1 = 1.37, 95% CI: 1.21, 1.55), and stroke (HRQ4 vs. Q1 = 1.16, 95% CI: 1.01, 1.33) but not with deaths due to injury, diabetes, or infectious disease. These results suggest that UVR exposure might not be beneficial for longevity.
doi:10.1093/aje/kws589
PMCID: PMC3736750  PMID: 23863757
epidemiology; mortality; prospective; sunlight; ultraviolet radiation; vitamin D
8.  Modeling epilepsy disparities among ethnic groups in Philadelphia, PA 
Statistics in medicine  2008;27(20):4069-4085.
SUMMARY
The Centers for Disease Control and Prevention defined epilepsy as an emerging public health issue in a recent report and emphasized the importance of epilepsy studies in minorities and people of low socioeconomic status. Previous research has suggested that the incidence rate for epilepsy is positively associated with various measures of social and economic disadvantage. In response, we utilize hierarchical Bayesian models to analyze health disparities in epilepsy and seizure risks among multiple ethnicities in the city of Philadelphia, Pennsylvania. The goals of the analysis are to highlight any overall significant disparities in epilepsy risks between the populations of Caucasians, African Americans, and Hispanics in the study area during the years 2002–2004 and to visualize the spatial pattern of epilepsy risks by ethnicity to indicate where certain ethnic populations were most adversely affected by epilepsy within the study area. Results of the Bayesian model indicate that Hispanics have the highest epilepsy risk overall, followed by African Americans, and then Caucasians. There are significant increases in relative risk for both African Americans and Hispanics when compared with Caucasians, as indicated by the posterior mean estimates of 2.09 with a 95 per cent credible interval of (1.67, 2.62) for African Americans and 2.97 with a 95 per cent credible interval of (2.37, 3.71) for Hispanics. Results also demonstrate that using a Bayesian analysis in combination with geographic information system (GIS) technology can reveal spatial patterns in patient data and highlight areas of disparity in epilepsy risk among subgroups of the population.
doi:10.1002/sim.3261
PMCID: PMC4106683  PMID: 18381676
hierarchical Bayesian regression; health disparities; spatial epidemiology; MCMC; spatial statistics
9.  A Primer on a Hepatocellular Carcinoma Bioresource Bank Using the Cancer Genome Atlas Guidelines: Practical Issues and Pitfalls 
World journal of surgery  2011;35(8):1732-1737.
Since the advent of the human genome, the era of personalized genomic medicine is indisputably in progress. In an effort to contribute to the evolving knowledge of genomic medicine, we aim directly at building a bioresource bank for hepatocellular carcinoma. This tumor bank is based on the rigorous guidelines set forth by the National Cancer Institute, and offers analytes to help elucidate the mechanisms of progression from cirrhosis to malignancy, risk factors for recurrence, and applicability of current treatment options to a diverse group of people. Surgeons have a privileged position between the patient (and their cancer) and the benches of basic science. Thus, we offer a primer based on our own experiences for which the surgeon may take to build their own bioresource bank and use to collaborate with others. We highlight some practicalities and pitfalls that could be overlooked, as well as discussion of solutions.
doi:10.1007/s00268-010-0953-y
PMCID: PMC4083491  PMID: 21221581
Bioresource bank; Biospecimen repository; The Cancer Genome Atlas; Translational research; Hepatocellular Carcinoma
10.  Integrative genomic characterization of oral squamous cell carcinomaidentifies frequent somatic drivers 
Cancer discovery  2013;3(7):10.1158/2159-8290.CD-12-0537.
The survival of patients with oral squamous cell carcinoma (OSCC) has not changed significantly in several decades, leading clinicians and investigators to search for promising molecular targets. To this end, we performed comprehensive genomic analysis of gene expression, copy number, methylation and point mutations in OSCC. Integrated analysis revealed more somatic events than previously reported, identifying four major driver pathways (mitogenic signaling, Notch, cell cycle, TP53) and two additional key genes (FAT1, CASP8). The Notch pathway was defective in 66% of patients, and in follow-up studies of mechanism, functional NOTCH1 signaling inhibited proliferation of OSCC cell lines. Frequent mutation of CASP8 defines a new molecular subtype of OSCC with few copy number changes. Although genomic alterations are dominated by loss of tumor suppressor genes, 80% of patients harbored at least one genomic alteration in a targetable gene, suggesting that novel approaches to treatment may be possible for this debilitating disease.
doi:10.1158/2159-8290.CD-12-0537
PMCID: PMC3858325  PMID: 23619168
Integrated genomics; head and neck/oral cancers; NOTCH1; CASP8
11.  PEX7 and EBP50 Target iNOS to the Peroxisome in Hepatocytes 
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.
doi:10.1016/j.niox.2013.02.084
PMCID: PMC3642215  PMID: 23474170
Inflammation; Sepsis; Inducible Nitric Oxide Synthase; Peroxisome; Liver; Subcellular Localization
12.  Whole-Genome Sequencing in a Patient with Charcot–Marie–Tooth Neuropathy 
The New England journal of medicine  2010;362(13):1181-1191.
BACKGROUND
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.
METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
doi:10.1056/NEJMoa0908094
PMCID: PMC4036802  PMID: 20220177
13.  Comparison of Algorithm-based Estimates of Occupational Diesel Exhaust Exposure to Those of Multiple Independent Raters in a Population-based Case–Control Study 
Annals of Occupational Hygiene  2012;57(4):470-481.
Objectives:
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.
Methods:
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.
Results:
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.
Discussion:
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.
doi:10.1093/annhyg/mes082
PMCID: PMC3629988  PMID: 23184256
case–control; diesel exhaust; expert judgement; exposure assessment
14.  Feasibility of a Clinical Trial of Vision Therapy for Treatment of Amblyopia 
Purpose
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1097/OPX.0b013e31828def04
PMCID: PMC3662294  PMID: 23563444
amblyopia; patching; vision therapy; placebo vision therapy; masking
15.  Inside the black box: starting to uncover the underlying decision rules used in one-by-one expert assessment of occupational exposure in case-control studies 
Objectives
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.
Methods
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.
Results
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%).
Conclusions
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.
doi:10.1136/oemed-2012-100918
PMCID: PMC3975600  PMID: 23155187
diesel exhaust; classification; data mining; occupational exposure
16.  Identification of a recurrent germline PAX5 mutation and susceptibility to pre-B cell acute lymphoblastic leukemia 
Nature genetics  2013;45(10):1226-1231.
doi:10.1038/ng.2754
PMCID: PMC3919799  PMID: 24013638
17.  Identification of TP53 as an Acute Lymphocytic Leukemia Susceptibility Gene Through Exome Sequencing 
Pediatric blood & cancer  2012;60(6):E1-E3.
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.
doi:10.1002/pbc.24417
PMCID: PMC3926299  PMID: 23255406
exome sequencing; acute lymphocytic leukemia; genetic predisposition to disease; genetic testing
18.  MLH1-Silenced and Non-Silenced Subgroups of Hypermutated Colorectal Carcinomas Have Distinct Mutational Landscapes 
The Journal of pathology  2013;229(1):99-110.
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.
doi:10.1002/path.4087
PMCID: PMC3926301  PMID: 22899370
colorectal cancer; microsatellite instability; MLH1; APC; KRAS; BRAF; WNT signaling; mutation rate
19.  Combined sequence-based and genetic mapping analysis of complex traits in outbred rats 
Nature genetics  2013;45(7):10.1038/ng.2644.
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.
doi:10.1038/ng.2644
PMCID: PMC3821058  PMID: 23708188
20.  Obligate mutualism within a host drives the extreme specialization of a fig wasp genome 
Genome Biology  2013;14(12):R141.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/gb-2013-14-12-r141
PMCID: PMC4053974  PMID: 24359812
21.  Vector Contact Rates on Eastern Bluebird Nestlings Do Not Indicate West Nile Virus Transmission in Henrico County, Virginia, USA 
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.
doi:10.3390/ijerph10126366
PMCID: PMC3881119  PMID: 24287858
host-seeking rate; nestling; nest mosquito trap; arbovirus; West Nile virus
22.  Function and Evolution of DNA Methylation in Nasonia vitripennis 
PLoS Genetics  2013;9(10):e1003872.
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.
Author Summary
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.
doi:10.1371/journal.pgen.1003872
PMCID: PMC3794928  PMID: 24130511
23.  Comparison of two expert-based assessments of diesel exhaust exposure in a case-control study: Programmable decision rules versus expert review of individual jobs 
Objectives
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1136/oemed-2011-100524
PMCID: PMC3439531  PMID: 22843440
24.  Prospective study of ultraviolet radiation exposure and risk of cancer in the U.S. 
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.
doi:10.1002/ijc.27619
PMCID: PMC3402606  PMID: 22539073
Ultraviolet radiation; cancer; vitamin D; prospective
25.  Spatial-temporal analysis of non-Hodgkin lymphoma risk using multiple residential locations 
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.
doi:10.1016/j.sste.2012.04.009
PMCID: PMC3372929  PMID: 22682442
cancer; generalized additive model; spatial risk; latency; exposure

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