Grier P. Page

Microarray data analysis: from disarray to consolidation and consensus

In just a few years, microarrays have gone from obscurity to being almost ubiquitous in biological research. At the same time, the statistical methodology for microarray analysis has progressed from simple visual assessments of results to a weekly deluge of papers that describe purportedly novel algorithms for analysing changes in gene expression. Although the many procedures that are available might be bewildering to biologists who wish to apply them, statistical geneticists are recognizing commonalities among the different methods. Many are special cases of more general models, and points of consensus are emerging about the general approaches that warrant use and elaboration.

Repeatability of published microarray gene expression analyses

Given the complexity of microarray-based gene expression studies, guidelines encourage transparent design and public data availability. Several journals require public data deposition and several public databases exist. However, not all data are publicly available, and even when available, it is unknown whether the published results are reproducible by independent scientists. Here we evaluated the replication of data analyses in 18 articles on microarray-based gene expression profiling published in Nature Genetics in 2005–2006. One table or figure from each article was independently evaluated by two teams of analysts. We reproduced two analyses in principle and six partially or with some discrepancies; ten could not be reproduced. The main reason for failure to reproduce was data unavailability, and discrepancies were mostly due to incomplete data annotation or specification of data processing and analysis. Repeatability of published microarray studies is apparently limited. More strict publication rules enforcing public data availability and explicit description of data processing and analysis should be considered.

Identification of a gene expression signature associated with recurrent disease in squamous cell carcinoma of the head and neck.

Molecular studies of squamous cell carcinoma of the head and neck (HNSCC) have demonstrated multiple genetic abnormalities such as activation of various oncogenes (Ras, Myc, epidermal growth factor receptor, and cyclin D1), tumor suppressor gene inactivation (TP53 and p16), and loss of heterozygosity at numerous chromosomal locations. Despite these observations, accurate and reliable biomarkers that predict patients at highest risk for local recurrence have yet to be defined. In an effort to identify gene expression signatures that may serve as biomarkers, we studied 41 squamous cell carcinoma tumors (25 primary and 16 locally recurrent) from various anatomical sites and 13 normal oral mucosal biopsy samples from healthy volunteers with microarray analysis using Affymetrix U133A GeneChip arrays. Differentially expressed genes were identified by calculating generalized t tests (P < 0.001) and applying a series of filtering criteria to yield a highly discriminant list of 2890 genes. Hierarchical clustering and image generation using standard software were used to visualize gene expression signatures. Several gene expression signatures were readily identifiable in the HNSCC tumors, including signatures associated with proliferation, extracellular matrix production, cytokine/chemokine expression, and immune response. Of particular interest was the association of a gene expression signature enriched for genes involved in tumor invasion and metastasis with patients experiencing locally recurrent disease. Notably, these tumors also demonstrated a marked absence of an immune response signature suggesting that modulation of tumor-specific immune responses may play a role in local treatment failure. These data provide evidence for a new gene expression-based biomarker of local treatment failure in HNSCC.

FLOWERING LOCUS T duplication coordinates reproductive and vegetative growth in perennial poplar

Annual plants grow vegetatively at early developmental stages and then transition to the reproductive stage, followed by senescence in the same year. In contrast, after successive years of vegetative growth at early ages, woody perennial shoot meristems begin repeated transitions between vegetative and reproductive growth at sexual maturity. However, it is unknown how these repeated transitions occur without a developmental conflict between vegetative and reproductive growth. We report that functionally diverged paralogs FLOWERING LOCUS T1 (FT1) and FLOWERING LOCUS T2 (FT2), products of whole-genome duplication and homologs of Arabidopsis thaliana gene FLOWERING LOCUS T (FT), coordinate the repeated cycles of vegetative and reproductive growth in woody perennial poplar (Populus spp.). Our manipulative physiological and genetic experiments coupled with field studies, expression profiling, and network analysis reveal that reproductive onset is determined by FT1 in response to winter temperatures, whereas vegetative growth and inhibition of bud set are promoted by FT2 in response to warm temperatures and long days in the growing season. The basis for functional differentiation between FT1 and FT2 appears to be expression pattern shifts, changes in proteins, and divergence in gene regulatory networks. Thus, temporal separation of reproductive onset and vegetative growth into different seasons via FT1 and FT2 provides seasonality and demonstrates the evolution of a complex perennial adaptive trait after genome duplication.

Familial and Genetic Susceptibility to Major Neonatal Morbidities in Preterm Twins

BACKGROUND. Intraventricular hemorrhage, necrotizing enterocolitis, and bronchopulmonary dysplasia remain significant causes of morbidity and mortality in preterm newborns. OBJECTIVES. Our goal was to assess the familial and genetic susceptibility to intraventricular hemorrhage, necrotizing enterocolitis, and bronchopulmonary dysplasia. METHODS. Mixed-effects logistic-regression and latent variable probit model analysis were used to assess the contribution of several covariates in a multicenter retrospective study of 450 twin pairs born at ≤32 weeks of gestation. To determine the genetic contribution, concordance rates in a subset of 252 monozygotic and dizygotic twin pairs were compared. RESULTS. The study population had a mean gestational age of 29 weeks and birth weight of 1286 g. After controlling for effects of covariates, the twin data showed that 41.3%, 51.9%, and 65.2%, respectively, of the variances in liability for intraventricular hemorrhage, necrotizing enterocolitis, and bronchopulmonary dysplasia could be accounted for by genetic and shared environmental factors. Among the 63 monozygotic twin pairs, the observed concordance for bronchopulmonary dysplasia was significantly higher than the expected concordance; 12 of 18 monozygotic twin pairs with ≥1 affected member had both members affected versus 3.69 expected. After controlling for covariates, genetic factors accounted for 53% of the variance in liability for bronchopulmonary dysplasia. CONCLUSIONS. Twin analyses show that intraventricular hemorrhage, necrotizing enterocolitis, and bronchopulmonary dysplasia are familial in origin. These data demonstrate, for the first time, the significant genetic susceptibility for bronchopulmonary dysplasia in preterm infants.

Inhaled NO accelerates restoration of liver function in adults following orthotopic liver transplantation

Ischemia/reperfusion (IR) injury in transplanted livers contributes to organ dysfunction and failure and is characterized in part by loss of NO bioavailability. Inhalation of NO is nontoxic and at high concentrations (80 ppm) inhibits IR injury in extrapulmonary tissues. In this prospective, blinded, placebo-controlled study, we evaluated the hypothesis that administration of inhaled NO (iNO; 80 ppm) to patients undergoing orthotopic liver transplantation inhibits hepatic IR injury, resulting in improved liver function. Patients were randomized to receive either placebo or iNO (n = 10 per group) during the operative period only. When results were adjusted for cold ischemia time and sex, iNO significantly decreased hospital length of stay, and evaluation of serum transaminases (alanine transaminase, aspartate aminotransferase) and coagulation times (prothrombin time, partial thromboplastin time) indicated that iNO improved the rate at which liver function was restored after transplantation. iNO did not significantly affect changes in inflammatory markers in liver tissue 1 hour after reperfusion but significantly lowered hepatocyte apoptosis. Evaluation of circulating NO metabolites indicated that the most likely candidate transducer of extrapulmonary effects of iNO was nitrite. In summary, this study supports the clinical use of iNO as an extrapulmonary therapeutic to improve organ function following transplantation.

Transcriptional coordination of the metabolic network in Arabidopsis

Patterns of coexpression can reveal networks of functionally related genes and provide deeper understanding of processes requiring multiple gene products. We performed an analysis of coexpression networks for 1,330 genes from the AraCyc database of metabolic pathways in Arabidopsis (Arabidopsis thaliana). We found that genes associated with the same metabolic pathway are, on average, more highly coexpressed than genes from different pathways. Positively coexpressed genes within the same pathway tend to cluster close together in the pathway structure, while negatively correlated genes typically occupy more distant positions. The distribution of coexpression links per gene is highly skewed, with a small but significant number of genes having numerous coexpression partners but most having fewer than 10. Genes with multiple connections (hubs) tend to be single-copy genes, while genes with multiple paralogs are coexpressed with fewer genes, on average, than single-copy genes, suggesting that the network expands through gene duplication, followed by weakening of coexpression links involving duplicate nodes. Using a network-analysis algorithm based on coexpression with multiple pathway members (pathway-level coexpression), we identified and prioritized novel candidate pathway members, regulators, and cross pathway transcriptional control points for over 140 metabolic pathways. To facilitate exploration and analysis of the results, we provide a Web site (http://www.transvar.org/at_coexpress/analysis/web) listing analyzed pathways with links to regression and pathway-level coexpression results. These methods and results will aid in the prioritization of candidates for genetic analysis of metabolism in plants and contribute to the improvement of functional annotation of the Arabidopsis genome.

Adipose tissue energy metabolism: altered gene expression profile of mice subjected to long-term caloric restriction

We investigated the influences of short‐term and lifespan‐prolonging long‐term caloric restriction (LCR) on gene expression in white adipose tissue (WAT). Over 11,000 genes were examined using high‐density oligonucleotide microarrays in four groups of 10‐to 11‐month‐old male C57Bl6 mice that were either fasted for 18 h before death (F), subjected to short‐term caloric restriction for 23 days (SCR), or LCR for 9 months and compared with nonfasted control (CO) mice. Only a few transcripts of F and SCR were differentially expressed compared with CO mice. In contrast, 345 transcripts of 6,266 genes found to be expressed in WAT were altered significantly by LCR. The expression of several genes encoding proteins involved in energy metabolism was increased by LCR. Further, many of the shifts in gene expression after LCR are known to occur during adipocyte differentiation. Selected LCR‐associated alterations of gene expression were supported by quantitative reverse transcriptase‐polymerase chain reaction, histology, and histochemical examinations. Our data provide new insights on the metabolic state associated with aging retardation by LCR.

Modification of the Mitochondrial Proteome in Response to the Stress of Ethanol-dependent Hepatotoxicity

Mitochondria are particularly susceptible to increased formation of reactive oxygen and nitrogen species in the cell that can occur in response to pathological and xenobiotic stimuli. Proteomics can give insights into both mechanism of pathology and adaptation to stress. Herein we report the use of proteomics to evaluate alterations in the levels of mitochondrial proteins following chronic ethanol exposure in an animal model. Forty-three proteins showed differential expression, 13 increased and 30 decreased, as a consequence of chronic ethanol. Of these proteins, 25 were not previously known to be affected by chronic ethanol emphasizing the power of proteomic approaches in revealing global responses to stress. Both nuclear and mitochondrially encoded gene products of the oxidative phosphorylation complexes in mitochondria from ethanol-fed rats were decreased suggesting an assembly defect in this integrated metabolic pathway. Moreover mtDNA damage was increased by ethanol demonstrating that the effects of ethanol consumption extend beyond the proteome to encompass mtDNA. Taken together, we have demonstrated that chronic ethanol consumption extends to a modification of the mitochondrial proteome far broader than realized previously. These data also suggest that the response of mitochondria to stress may not involve non-discriminate changes in the proteome but is restricted to those metabolic pathways that have a direct role in a specific pathology.

MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors

Neurofibromatosis type 1 (NF1) patients develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). These incurable peripheral nerve tumors result from loss of NF1 tumor suppressor gene function, causing hyperactive Ras signaling. Activated Ras controls numerous downstream effectors, but specific pathways mediating the effects of hyperactive Ras in NF1 tumors are unknown. We performed cross-species transcriptome analyses of mouse and human neurofibromas and MPNSTs and identified global negative feedback of genes that regulate Ras/Raf/MEK/ERK signaling in both species. Nonetheless, ERK activation was sustained in mouse and human neurofibromas and MPNST. We used a highly selective pharmacological inhibitor of MEK, PD0325901, to test whether sustained Ras/Raf/MEK/ERK signaling contributes to neurofibroma growth in a neurofibromatosis mouse model (Nf1(fl/fl);Dhh-Cre) or in NF1 patient MPNST cell xenografts. PD0325901 treatment reduced aberrantly proliferating cells in neurofibroma and MPNST, prolonged survival of mice implanted with human MPNST cells, and shrank neurofibromas in more than 80% of mice tested. Our data demonstrate that deregulated Ras/ERK signaling is critical for the growth of NF1 peripheral nerve tumors and provide a strong rationale for testing MEK inhibitors in NF1 clinical trials.