Comprehensive, Integrative Genomic Analysis of Diffuse Lower-Grade Gliomas.

BACKGROUND: Diffuse low-grade and intermediate-grade gliomas (which together make up the lower-grade gliomas, World Health Organization grades II and III) have highly variable clinical behavior that is not adequately predicted on the basis of histologic class. Some are indolent; others quickly progress to glioblastoma. The uncertainty is compounded by interobserver variability in histologic diagnosis. Mutations in IDH, TP53, and ATRX and codeletion of chromosome arms 1p and 19q (1p/19q codeletion) have been implicated as clinically relevant markers of lower-grade gliomas. METHODS: We performed genomewide analyses of 293 lower-grade gliomas from adults, incorporating exome sequence, DNA copy number, DNA methylation, messenger RNA expression, microRNA expression, and targeted protein expression. These data were integrated and tested for correlation with clinical outcomes. RESULTS: Unsupervised clustering of mutations and data from RNA, DNA-copy-number, and DNA-methylation platforms uncovered concordant classification of three robust, nonoverlapping, prognostically significant subtypes of lower-grade glioma that were captured more accurately by IDH, 1p/19q, and TP53 status than by histologic class. Patients who had lower-grade gliomas with an IDH mutation and 1p/19q codeletion had the most favorable clinical outcomes. Their gliomas harbored mutations in CIC, FUBP1, NOTCH1, and the TERT promoter. Nearly all lower-grade gliomas with IDH mutations and no 1p/19q codeletion had mutations in TP53 (94%) and ATRX inactivation (86%). The large majority of lower-grade gliomas without an IDH mutation had genomic aberrations and clinical behavior strikingly similar to those found in primary glioblastoma. CONCLUSIONS: The integration of genomewide data from multiple platforms delineated three molecular classes of lower-grade gliomas that were more concordant with IDH, 1p/19q, and TP53 status than with histologic class. Lower-grade gliomas with an IDH mutation either had 1p/19q codeletion or carried a TP53 mutation. Most lower-grade gliomas without an IDH mutation were molecularly and clinically similar to glioblastoma. (Funded by the National Institutes of Health.).

Differential expression of pancreatitis-associated protein and thrombospondins in arterial versus venous tissues.

BACKGROUND/AIMS: Arteries and veins modulate cardiovascular homeostasis and contribute to hypertension pathogenesis. Functional differences between arteries and veins are based upon differences in gene expression. To better characterize these expression patterns, and to identify candidate genes that could be manipulated selectively in the venous system, we performed whole genome expression profiling of arteries and veins. METHODS: We used the CodeLink platform and the major artery (thoracic aorta) and vein (caudal vena cava) of the rat. RESULTS: The most prominent difference was pancreatitis-associated protein (PAP1), expressed 64-fold higher in vena cava versus aorta. Expression of mRNA for thrombospondins (TSP-1, TSP-4) was greater than 5-fold higher in veins versus arteries. Higher mRNA expression of TSP-1, TSP-2, TSP-4 and PAP1 in vena cava versus aorta was confirmed by PCR. Immunohistochemical analysis of tissue sections qualitatively confirmed a higher expression of these proteins in vena cava versus aorta. CONCLUSION: This is the first gene array study of adult rat arterial and venous tissues, and also the first study to report differences in inflammatory genes between arteries and veins. Data from these studies may provide novel insights into the genetic basis for functional differences between arteries and veins in health and disease.

So many studies, too few subjects: establishing functional relevance of genetic polymorphisms on pharmacokinetics.

Based on current literature, greater clarity in defining the magnitude of polymorphism effects on pharmacokinetics can be achieved by addressing key components of study design, including adequate subject numbers per study group. Convincing evidence of functional relevance exists for polymorphisms in genes such as CYP2D6 and UGT1A1, whereas the published evidence for similar effects for CYP3A5, OATP1B1, and ABCB1 is still emerging or equivocal. Polymorphism-associated differences in pharmacokinetic parameters were simulated to incorporate (1) the ratio of group mean parameter values for homozygous wild-type subjects versus homozygous variants, (2) pharmacokinetic variability, and (3) sample size needed to achieve 80% power, assuming 69% coefficient of variation. Subject selection by genotype and choice of probe substrate are also considered. Simulation results and literature examples are incorporated to define key recommendations for future investigations. This will allow for more definitive statements in publications regarding genotype influence on pharmacokinetics.

Neutrophil C5a receptor and the outcome in a rat model of sepsis.

Complement fragment 5a (C5a)-C5a receptor (C5aR) signaling plays an essential role in neutrophil innate immunity. Blockade of either the ligand or the receptor improves survival rates in experimental sepsis. In the current study, sepsis was induced in rats by cecal ligation/puncture. Early in sepsis C5aR content on neutrophils significantly dropped, reached the nadir at 24 h after onset of sepsis, and progressively elevated thereafter. Western-blot, RT-PCR, and confocal microscopy analyses revealed that the loss and re-expression of C5aR during sepsis might be due, at least in part, to the receptor internalization and reconstitution. The reduction and reconstitution of C5aR correlate with the loss and restoration of innate immune functions of blood neutrophils (chemotaxis and reactive oxygen species production), respectively. Quantitative measurements of C5aR on blood neutrophils are highly predictive of survival or death during sepsis. These data suggest that neutrophil C5aR content represents an essential component of an efficient defense system in sepsis and may serve as a prognostic marker for the outcome.

The upper airway response to pollen is enhanced by exposure to combustion particulates: a pilot human experimental challenge study.

Although human experimental studies have shown that gaseous pollutants enhance the inflammatory response to allergens, human data on whether combustion particulates enhance the inflammatory response to allergen are limited. Therefore, we conducted a human experimental study to investigate whether combustion particulates enhance the inflammatory response to aeroallergens. "Enhancement" refers to a greater-than-additive response when combustion particulates are delivered with allergen, compared with the responses when particulates and allergen are delivered alone. Eight subjects, five atopic and three nonatopic, participated in three randomized exposure-challenge sessions at least 2 weeks apart (i.e., clean air followed by allergen, particles followed by no allergen, or particles followed by allergen). Each session consisted of nasal exposure to combustion particles (target concentration of 1.0 mg/m3) or clean air for 1 hr, followed 3 hr later by challenge with whole pollen grains or placebo. Nasal lavage was performed immediately before particle or clean air exposure, immediately after exposure, and 4, 18 and 42 hr after pollen challenge. Cell counts, differentials, and measurement of cytokines were performed on each nasal lavage. In atopic but not in nonatopic subjects, when allergen was preceded by particulates, there was a significant enhancement immediately after pollen challenge in nasal lavage leukocytes and neutrophils (29.7 X 10(3) cells/mL and 25.4 X 10(3) cells/mL, respectively). This represents a 143% and 130% enhancement, respectively. The enhanced response for interleukin-4 was 3.23 pg/mL (p = 0.06), a 395% enhancement. In atopic subjects there was evidence of an enhanced response when particulates, as compared to clean air, preceded the allergen challenge.

Expression and regulation of the macrophage inflammatory protein-1 alpha gene by nicotine in rat alveolar macrophages.

Cigarette smoking causes inflammation mainly confined to the airway and lung. Nicotine is one of the primary constituents in cigarette smoke. Alveolar macrophages apparently play a pivotal role in mediating pulmonary inflammation via the production of chemokines. Macrophage inflammatory protein-1 alpha (MIP-1 alpha), a member of CC chemokines, has been shown to contribute to monocyte/macrophage and neutrophil chemotaxis and activation. Our previous work demonstrated that MIP-1 alpha mRNA expression in macrophages is induced by a variety of stimuli. In the present study, we further investigate whether nicotine can regulate the gene expression of MIP-1 alpha in macrophages and determine the mechanism leading to increased expression. A rat alveolar macrophage (RAM) cell line, NR8383, was treated with nicotine at a dose of 3.1, 31, 310 microM, or 3.1 mM. Northern blot analysis showed that the induction of MIP-1 alpha mRNA expression was dose-dependent. To define the time course of the inflammatory response, RAM cells were exposed to 31 microM nicotine, MIP-1 alpha mRNA was induced as early as 1 h after treatment, was maximally expressed at 4 and 6 hours, and reduced by 8 hours. Western blot analysis demonstrated a single band with an estimated molecular weight of 10 kD for MIP-1 alpha which was induced after nicotine treatment, suggesting that expression of MIP-1 alpha mRNA could reflect in protein synthesis. In addition. the increase in MIP-1 alpha mRNA expression induced by nicotine was attenuated by co-treatment with the antioxidant N-acetylcysteine (NAC), at doses of 10 and 20 mM, suggesting that the induction of MIP-1 alpha mRNA is mediated via the generation of reactive oxygen species (ROS). To further investigate transcriptional regulation of the MIP-1 alpha gene expression, RAM cells were exposed to nicotine. MIP-1 alpha mRNA levels were significantly increased in nuclear RNA preparations, indicating that transcriptional activation is involved in increased expression of MIP-1 alpha mRNA. Moreover, we performed RNA decay assay by measuring the half-life of MIP-1 alpha mRNA. Treatment of RAM cells with the transcriptional inhibitor actinomycin D following exposure to nicotine revealed that the half-life of MIP-1 alpha mRNA was markedly increased by nicotine treatment, supporting a role of post-transcriptional stabilization in MIP-1 alpha gene expression. These observations indicate that nicotine can induce MIP-1 alpha mRNA expression and protein synthesis in RAM cells, mediating, at least in part, via the generation of ROS. In addition, the increase in MIP-1 alpha mRNA level involves, both transcriptional activation and post-transcriptional stabilization.

Association between human paraoxonase gene polymorphism and chronic symptoms in pesticide-exposed workers.

Pesticides, such as parathion, are metabolized by cytochrome p-450 system to paraoxon, which is a potent cholinesterase inhibitor. Paraoxonase (PON) catalyzes the hydrolysis of these toxic metabolites and protects against pesticide toxicity. A glutamine/arginine (Gln/Arg) polymorphism at amino acid position 192 of PON has been described. The Arg/Arg genotype is associated with higher serum paraoxonase activity compared to Gln/Gln. The Arg/Gln genotype is associated with intermediate serum PON activity. The potential association between PON genotype and symptoms of chronic pesticide toxicity was examined among 100 farm workers. As part of a cross-sectional study of pesticide toxicity among mixed-race farm workers in the Western Cape. South Africa, 100 farm workers were genotyped for polymorphism of the paraoxonase gene at amino acid position 192. Subjects with two or more of the following symptoms were considered to have evidence of chronic toxicity: abdominal pain, nausea, rhinorrhea, dizziness, headache, somnolence, fatigue, gait disturbance, limb numbness, paresthesias, limb pain, or limb weakness. In multivariable logistic regression analysis, the independent predictors of chronic toxicity were previous history of head trauma resulting in loss of consciousness (OR 2.8, 95% CI = 1.7-6.7), having worked as a pesticide applicator (OR 5.4, 95% CI = 3.2-8.9), and having one of the two "slow metabolism" (Gln/Gln or Gln/Arg) genotypes (OR 2.9, 95% CI = 1.7-6.9). Furthermore, the prevalence of chronic toxicity increased in a stepwise fashion from 15% among pesticide nonapplicators with a "fast metabolism" (Arg/Arg) genotype, to 42.9% among pesticide nonapplicators with "slow metabolism" (Gln/Gln or Gln/Arg) genotypes, to 58.8% among pesticide applicators with "fast metabolism" genotype, and 75.0% among pesticide applicators with "slow metabolism" genotypes (P = 0.001). Age, number of years on the job, smoking history, alcohol history, education level, plasma or red blood cell cholinesterase level, or previous history of acute organophosphate poisoning were not statistically significant predictors of chronic toxicity. The PON genotype is an important determinant of a farmworker's susceptibility to chronic pesticide poisoning.

Contamination of water supplies with Cryptosporidium parvum and Giardia lamblia and diarrheal illness in selected Russian cities.

Cryptosporidium parvum and Giardia lamblia are important agents of waterborne diarrheal illness worldwide. While giardiasis is routinely diagnosed in Russia with a chemical staining technique, data on the prevalence of cryptosporidiosis are scarce. Monitoring of the respective parasites in water supplies in Russia is very limited. A health survey conducted in the city of Cherepovets and three other cities in the European part of Russia using enzyme-linked immunosorbent assays (ELISA) demonstrated that 6.9% of diarrheal patients tested had C. parvum antigens in their fecal samples; 9.4% had G. lamblia antigens. A survey of occurrence of these parasites in water supplies in Cherepovets and seven other cities demonstrated that source and finished water samples from several of these cities were contaminated with either C. parvum oocysts or G. lamblia cysts. The surveys were not designed to assess associations between presence or concentrations of C. parvum and G. lamblia in water and related gastrointestinal diseases in exposed populations. Rather, the goals were to demonstrate the presence of disinfection-resistant protozoan parasites in untreated and treated waters, and the importance of these pathogens as causative agents of diarrheal illnesses in a number of Russian cities.

Two algorithms for biospecimen comparison and differentiation using SNP genotypes.

AIMS: Biobanks are frequently required to verify specimen relationships. We present two algorithms to compare SNP genotype patterns that provide an objective, high-throughput tool for verification. METHODS: The first algorithm allows for comparison of all holdings within a biobank, and is well suited to construct sample relationships de novo for comparison with assumed relationships. The second algorithm is tailored to oncology, and allows one to confirm that paired DNAs from malignant and normal tissues are from the same individual in the presence of copy number variations. To evaluate both algorithms, we used an internal training data set (n = 1504) and an external validation data set (n = 1457). RESULTS: In comparison with the results from manual review and a priori knowledge of patient relationships, we identified no errors in interpreting sample relationships within our validation data set. CONCLUSION: We provide an efficient and objective method of automated data analysis that is currently lacking for establishing and verifying specimen relationships in biobanks.

Opening the gate for genomics data into clinical research: a use case in managing patients' DNA samples from the bench to drug development.

The use of human genetic polymorphism data in drug development is not a recent event. Typically, the detection of patients' genetic variations in drug-metabolizing enzymes has become common practice in clinical laboratories. What is new is the scale and diversity of genomics data that has entered into the drug research and development decision-making process. At least three concurrent events contribute to this paradigm shift: first the growing body of evidence that establishes that interindividual variation in both therapeutic response and adverse events are attributable to a genetic component; second the technological progress that enables the consistent and reproducible detection of human genomic quantities; third the expectation that the productivity of new drug development will be increased by identifying which patients would benefit from candidate therapies early in the clinical process. This influx of human genomics data into clinical laboratories requires some logistical adjustment in terms of data management. The major specifications of an information solution system intended for a clinical genomic laboratory are its compliance with regulatory procedures, regarding the handling of human genetic data and its subsequent integration into an existing clinical data management system from the hosting institution. The purpose of this article is to inform the community of the challenges in setting up a center for genomics data that ensures accurate, traceable and integrated data for laboratory management. This is by no means the only way to accomplish the same goal, and is simply presented as one way that Pfizer chose to solve these issues.

Investigating fixative-induced changes in RNA quality and utility by microarray analysis.

Described herein is a detailed analysis of the impact of three fixatives (10% neutral buffered formalin, modified methacarn and 70% ethanol) on RNA quality and utility using microarray analysis compared to OCT-embedded and flash frozen tissue. From rat livers fixed and stored in paraffin blocks for 1 month or 1 year, RNA was isolated and applied to rat whole genome microarrays. At both time points, RNA isolated from OCT-embedded tissue lost up to 5% of the information contained in snap frozen control liver. Of the fixatives used, modified methacarn was associated with the smallest loss of RNA information content (approximately 10%), while liver fixed in 70% ethanol and 10% neutral buffered formalin lost roughly 25% and 80%, respectively. We conclude that when optimum morphology is required for techniques such as laser microdissection, modified methacarn is the fixative least harmful to nucleic acids of the three tested in this study. In contrast, using traditional isolation techniques, RNA derived from tissue fixed in 10% NBF will not give reliable results on microarray studies, and should be reserved for techniques less affected by the fragmentation and modification of the template RNA, such as quantitative RT-PCR.

Limited influence of UGT1A1*28 and no effect of UGT2B7*2 polymorphisms on UGT1A1 or UGT2B7 activities and protein expression in human liver microsomes.

AIMS: UGT1A1 and UGT2B7 are enzymes that commonly contribute to drug glucuronidation. Since genetic factors have been suggested to contribute to variability in activities and expression levels of these enzymes, a quantitative assessment of the influence of the major genotypes (UGT1A1*28 or UGT2B7*2) on enzyme activities was conducted. METHODS: Using a bank of microsomal samples from 59 human livers, the effect of UGT1A1*28 or UGT2B7*2 polymorphisms were investigated on rates of estradiol 3-glucuronidation (a marker of UGT1A1 enzyme activity) or zidovudine glucuronidation (a marker of UGT2B7 enzyme activity) and levels of immunoreactive protein for each enzyme. Glucuronidation rates for both enzymes were measured at K(m)/S(50) and 10 times K(m)/S(50) concentrations. RESULTS: UGT1A1 and UGT2B7 enzyme activities varied up to 16-fold and sixfold, respectively. Rates at K(m)/S(50) concentration closely correlated with rates at 10 times K(m)/S(50) concentration for both enzymes (but not at 1/10th K(m) for UGT2B7). Enzyme activities correlated with relative levels of immunoreactive protein for UGT1A1 and UGT2B7. Furthermore, rates of zidovudine glucuronidation correlated well with rates of glucuronidation of the UGT2B7 substrate gemcabene, but did not correlate with UGT1A1 enzyme activities. For the UGT1A1*28 polymorphism, consistent with levels of UGT1A1 immunoreactive protein, mean UGT1A1 activity was 2.5- and 3.2-fold lower for TA(6)/TA(7) (P < 0.05) and TA(7)/TA(7) (P < 0.001) genotypes in comparison with the TA(6)/TA(6) genotype. CONCLUSIONS: Relative to the observed 16-fold variability in UGT1A1 activity, these data indicate only a partial (approximately 40%) contribution of the UGT1A1*28 polymorphism to variability of interindividual differences in UGT1A1 enzyme activity. For the UGT2B7*2 polymorphism, genotype had no influence on immunoreactive UGT2B7 protein or the rate of 3'-azido-3'-deoxythymidine glucuronidation.

Assessment of fixatives, fixation, and tissue processing on morphology and RNA integrity.

Molecular characterization of morphologic change requires exquisite tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses on fixed tissues, we assessed morphologic and RNA integrity in rat liver when sections were fixed in 70% neutral-buffered formalin, modified Davidson's II, 70% ethanol, UMFIX, modified Carnoy's, modified methacarn, Bouin's, phosphate-buffered saline, or 30% sucrose. Each sample was subjected to standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity. Modified methacarn, 70% ethanol, and modified Carnoy's solution each resulted in tissue morphology representing a reasonable alternative to formalin. Modified methacarn and UMFIX best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was modestly improved. We conclude that modified methacarn, 70% ethanol, and modified Carnoy's solution provided acceptable preservation of tissue morphology and RNA quality using both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens.

Altered neutrophil trafficking during sepsis.

In sepsis, dysregulation of the inflammatory system is well known, as reflected in excessive inflammatory mediator production, complement activation, and appearance of defects in phagocytic cells. In the current study sepsis was induced in rats by cecal ligation/puncture. Early in sepsis the beta(1) and beta(2) integrin content on blood neutrophils increased in a nontranscriptional manner, and the increase in beta(2), but not beta(1), integrin content was C5a dependent. Similar changes could be induced in vitro on blood neutrophils following contact with phorbol ester or C5a. Direct injury of lungs of normal rats induced by deposition of IgG immune complexes (IgG-IC) caused 5-fold increases in the myeloperoxidase content that was beta(2), but not beta(1), dependent. In contrast, in cecal ligation/puncture lungs myeloperoxidase increased 10-fold after IgG immune complex deposition and was both beta(1) and beta(2) integrin dependent. These data suggest that sepsis causes enhanced neutrophil trafficking into the lung via mechanisms that are not engaged in the nonseptic state.