Transcriptome analysis describing new immunity and defense genes in peripheral blood mononuclear cells of rheumatoid arthritis patients

Abstract

Background: Large-scale gene expression profiling of peripheral blood mononuclear cells from Rheumatoid Arthritis (RA) patients could provide a molecular description that reflects the contribution of diverse cellular responses associated with this disease. The aim of our study was to identify peripheral blood gene expression profiles for RA patients, using Illumina technology, to gain insights into RA molecular mechanisms.

Methodology/principal findings: The Illumina Human-6v2 Expression BeadChips were used for a complete genome-wide transcript profiling of peripheral blood mononuclear cells (PBMCs) from 18 RA patients and 15 controls. Differential analysis per gene was performed with one-way analysis of variance (ANOVA) and P values were adjusted to control the False Discovery Rate (FDR<5%). Genes differentially expressed at significant level between patients and controls were analyzed using Gene Ontology (GO) in the PANTHER database to identify biological processes. A differentially expression of 339 Reference Sequence genes (238 down-regulated and 101 up-regulated) between the two groups was observed. We identified a remarkably elevated expression of a spectrum of genes involved in Immunity and Defense in PBMCs of RA patients compared to controls. This result is confirmed by GO analysis, suggesting that these genes could be activated systemically in RA. No significant down-regulated ontology groups were found. Microarray data were validated by real time PCR in a set of nine genes showing a high degree of correlation.

Conclusions/significance: Our study highlighted several new genes that could contribute in the identification of innovative clinical biomarkers for diagnostic procedures and therapeutic interventions.

Figure 1. Scatter plots representation of signal intensities.

Scatter plot of technical replicates: (A) same reference RNA undergoing two different hybridization or (B) same reference RNA from two different labeling runs. Typical scatter plots of data obtained from two patients (C) or a patient and a control (D). Pearson correlation coefficient is indicated in each scatter plot.

Figure 2. Cluster diagram of the expression of 339 significantly expressed genes in 18 RA patients and 15 controls.

Genes are organized by hierarchical clustering based on overall similarity in expression patterns. Red represents relative expression greater than the median expression level across all samples, and green represents an expression level lower than the median. Black indicates intermediate expression.

Figure 3. Gene ontology analysis of the most representative biological processes represented by the 101 up-regulated genes in RA patients.

Gene Ontology (GO) analysis in the PANTHER database was applied for an interpretation of the biological processes that are represented by the genes showing a higher significantly different expression level in RA patients compared to the controls. A and B - Biological processes with a P value <0.05 (red bars) were considered significant after Bonferroni correction. The GO analysis P-value was plotted on the y axis versus biological processes on the x axis. A - The binomial statistics tool was used to compare our gene list to a reference list (NCBI: Homo sapiens genes) determining the statistically significant (P value) over- or under- representation of PANTHER biological process. B - The Mann-Whitney U Test was used to determine significant P values which indicate that the distribution (fold change) for each biological process is non-random and different from the overall distribution. OP - Oxidative Phosphorylation; ET - Electron Transport; ID - Immunity and Defense; NNNAM - Nucleoside, Nucleotide and Nucleic Acid Metabolism; T – Transport.

Figure 4. Correlation between microarray and real time PCR data.

The scatter plot compares mean expression of RA patients/controls ratio for nine genes. Each point represents the RA patients/controls ratio from the microarray (y axis) and real time PCR (x axis). Pearson correlation coefficient is indicated in the scatter plot.

Figure 5. Biological functions of Immunity and Defense genes highlighted in our Rheumatoid Arthritis study.

The genes differentially expressed in Immunity and Defense process are stratified in four functional related mechanisms: pro-inflammation, anti-microbial activity, oxidative and osmotic cellular stress and immunomodulatory functions.