Comparison of gene expression microarray data with count-based RNA measurements informs microarray interpretation

Background

Although numerous investigations have compared gene expression microarray platforms, preprocessing methods and batch correction algorithms using constructed spike-in or dilution datasets, there remains a paucity of studies examining the properties of microarray data using diverse biological samples. Most microarray experiments seek to identify subtle differences between samples with variable background noise, a scenario poorly represented by constructed datasets. Thus, microarray users lack important information regarding the complexities introduced in real-world experimental settings. The recent development of a multiplexed, digital technology for nucleic acid measurement enables counting of individual RNA molecules without amplification and, for the first time, permits such a study.

Results

Using a set of human leukocyte subset RNA samples, we compared previously acquired microarray expression values with RNA molecule counts determined by the nCounter Analysis System (NanoString Technologies) in selected genes. We found that gene measurements across samples correlated well between the two platforms, particularly for high-variance genes, while genes deemed unexpressed by the nCounter generally had both low expression and low variance on the microarray. Confirming previous findings from spike-in and dilution datasets, this “gold-standard” comparison demonstrated signal compression that varied dramatically by expression level and, to a lesser extent, by dataset. Most importantly, examination of three different cell types revealed that noise levels differed across tissues.

Conclusions

Microarray measurements generally correlate with relative RNA molecule counts within optimal ranges but suffer from expression-dependent accuracy bias and precision that varies across datasets. We urge microarray users to consider expression-level effects in signal interpretation and to evaluate noise properties in each dataset independently.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-649) contains supplementary material, which is available to authorized users.     

Bimodal signatures of germline methylation are linked with gene expression plasticity in the coral Acropora millepora

Background

In invertebrates, genes belonging to dynamically regulated functional categories appear to be less methylated than “housekeeping” genes, suggesting that DNA methylation may modulate gene expression plasticity. To date, however, experimental evidence to support this hypothesis across different natural habitats has been lacking.

Results

Gene expression profiles were generated from 30 pairs of genetically identical fragments of coral Acropora millepora reciprocally transplanted between distinct natural habitats for 3 months. Gene expression was analyzed in the context of normalized CpG content, a well-established signature of historical germline DNA methylation. Genes with weak methylation signatures were more likely to demonstrate differential expression based on both transplant environment and population of origin than genes with strong methylation signatures. Moreover, the magnitude of expression differences due to environment and population were greater for genes with weak methylation signatures.

Conclusions

Our results support a connection between differential germline methylation and gene expression flexibility across environments and populations. Studies of phylogenetically basal invertebrates such as corals will further elucidate the fundamental functional aspects of gene body methylation in Metazoa.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1109) contains supplementary material, which is available to authorized users.     

An Integrated Approach to Uncover Driver Genes in Breast Cancer Methylation Genomes

Background

Cancer cells typically exhibit large-scale aberrant methylation of gene promoters. Some of the genes with promoter methylation alterations play “driver” roles in tumorigenesis, whereas others are only “passengers”.

Results

Based on the assumption that promoter methylation alteration of a driver gene may lead to expression alternation of a set of genes associated with cancer pathways, we developed a computational framework for integrating promoter methylation and gene expression data to identify driver methylation aberrations of cancer. Applying this approach to breast cancer data, we identified many novel cancer driver genes and found that some of the identified driver genes were subtype-specific for basal-like, luminal-A and HER2+ subtypes of breast cancer.

Conclusion

The proposed framework proved effective in identifying cancer driver genes from genome-wide gene methylation and expression data of cancer. These results may provide new molecular targets for potential targeted and selective epigenetic therapy.     

Distance-based assessment of the localization of functional annotations in 3D genome reconstructions

Background

Recent studies used the contact data or three-dimensional (3D) genome reconstructions from Hi-C (chromosome conformation capture with next-generation sequencing) to assess the co-localization of functional genomic annotations in the nucleus. These analyses dichotomized data point pairs belonging to a functional annotation as “close” or “far” based on some threshold and then tested for enrichment of “close” pairs. We propose an alternative approach that avoids dichotomization of the data and instead directly estimates the significance of distances within the 3D reconstruction.

Results

We applied this approach to 3D genome reconstructions for Plasmodium falciparum, the causative agent of malaria, and Saccharomyces cerevisiae and compared the results to previous approaches. We found significant 3D co-localization of centromeres, telomeres, virulence genes, and several sets of genes with developmentally regulated expression in P. falciparum; and significant 3D co-localization of centromeres and long terminal repeats in S. cerevisiae. Additionally, we tested the experimental observation that telomeres form three to seven clusters in P. falciparum and S. cerevisiae. Applying affinity propagation clustering to telomere coordinates in the 3D reconstructions yielded six telomere clusters for both organisms.

Conclusions

Distance-based assessment replicated key findings, while avoiding dichotomization of the data (which previously yielded threshold-sensitive results).

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-992) contains supplementary material, which is available to authorized users.     

Reinterpreting Behavioral Receptive Fields: Lightness Induction Alters Visually Completed Shape

Background

A classification image (CI) technique has shown that static luminance noise near visually completed contours affects the discrimination of fat and thin Kanizsa shapes. These influential noise regions were proposed to reveal “behavioral receptive fields” of completed contours–the same regions to which early cortical cells respond in neurophysiological studies of contour completion. Here, we hypothesized that 1) influential noise regions correspond to the surfaces that distinguish fat and thin shapes (hereafter, key regions); and 2) key region noise biases a “fat” response to the extent that its contrast polarity (lighter or darker than background) matches the shape''s filled-in surface color.

Results

To test our hypothesis, we had observers discriminate fat and thin noise-embedded rectangles that were defined by either illusory or luminance-defined contours (Experiment 1). Surrounding elements (“inducers”) caused the shapes to appear either lighter or darker than the background–a process sometimes referred to as lightness induction. For both illusory and luminance-defined rectangles, key region noise biased a fat response to the extent that its contrast polarity (light or dark) matched the induced surface color. When lightness induction was minimized, luminance noise had no consistent influence on shape discrimination. This pattern arose when pixels immediately adjacent to the discriminated boundaries were excluded from the analysis (Experiment 2) and also when the noise was restricted to the key regions so that the noise never overlapped with the physically visible edges (Experiment 3). The lightness effects did not occur in the absence of enclosing boundaries (Experiment 4).

Conclusions

Under noisy conditions, lightness induction alters visually completed shape. Moreover, behavioral receptive fields derived in CI studies do not correspond to contours per se but to filled-in surface regions contained by those contours. The relevance of lightness to two-dimensional shape completion supplies a new constraint for models of object perception.     

FABP4 Dynamics in Obesity: Discrepancies in Adipose Tissue and Liver Expression Regarding Circulating Plasma Levels

Background

FABP4 is predominantly expressed in adipose tissue, and its circulating levels are linked with obesity and a poor atherogenic profile.

Objective

In patients with a wide BMI range, we analyze FABP4 expression in adipose and hepatic tissues in the settings of obesity and insulin resistance. Associations between FABP4 expression in adipose tissue and the FABP4 plasma level as well as the main adipogenic and lipolytic genes expressed in adipose tissue were also analyzed.

Methods

The expression of several lipogenic, lipolytic, PPAR family and FABP family genes was analyzed by real time PCR. FABP4 protein expression in total adipose tissues and its fractions were determined by western blot.

Results

In obesity FABP4 expression was down-regulated (at both mRNA and protein levels), with its levels mainly predicted by ATGL and inversely by the HOMA-IR index. The BMI appeared as the only determinant of the FABP4 variation in both adipose tissue depots. FABP4 plasma levels showed a significant progressive increase according to BMI but no association was detected between FABP4 circulating levels and SAT or VAT FABP4 gene expression. The gene expression of FABP1, FABP4 and FABP5 in hepatic tissue was significantly higher in tissue from the obese IR patients compared to the non-IR group.

Conclusion

The inverse pattern in FABP4 expression between adipose and hepatic tissue observed in morbid obese patients, regarding the IR context, suggests that both tissues may act in a balanced manner. These differences may help us to understand the discrepancies between circulating plasma levels and adipose tissue expression in obesity.     

Mutational Analysis of ATP8B1 in Patients with Chronic Pancreatitis

Background

Mutations in genes encoding cationic trypsinogen (PRSS1), pancreatic secretory trypsin inhibitor (SPINK1) and chymotrypsinogen C (CTRC) are associated with chronic pancreatitis. However, in many patients with a familial chronic pancreatitis pattern suggesting a genetic cause, no mutations in either of these genes can be found, indicating that other, still unknown, associated genes exist. In this respect ATP8B1 is an interesting candidate due to its strong expression in the pancreas, its supposed general function in membrane organization and the higher incidence of pancreatitis in patients with ATP8B1 deficiency.

Methods

We analyzed all 27 ATP8B1 coding exons and adjacent non-coding sequences of 507 chronic pancreatitis patients by direct sequencing. Exons that harbored possible relevant variations were subsequently sequenced in 1,027 healthy controls.

Results

In the exonic regions, 5 novel non-synonymous alterations were detected as well as 14 previously described alterations of which some were associated with ATP8B1 deficiency. However, allele frequencies for any of these variations did not significantly differ between patients and controls. Furthermore, several non-synonymous variants were exclusively detected in control subjects and multiple variants in the non-coding sequence were identified with similar frequencies in both groups.

Conclusions

We did not find an association between heterozygous ATP8B1 variants and chronic pancreatitis in our cohort of patients with hereditary and idiopathic chronic pancreatitis.