Application of genome-wide expression analysis to human health and disease

Abstract

The application of genome-wide expression analysis to a large-scale, multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition, variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject, among different healthy subjects, among different leukocyte subpopulations, and the effect of traumatic injury, were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast, genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly, traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects, and can be studied with confidence in human disease.

Figures

Fig. 1.

Variation in gene expression from healthy subjects and trauma patients. Blood leukocytes were obtained from four healthy subjects repeatedly over a 24-h study period, from 17 different healthy subjects, and from 14 patients after severe trauma. The coefficients of variation were determined at the probe set level and were plotted as a distribution curve.

Fig. 2.

Leukocyte populations in total white blood cells (WBCs) and T cell- and monocyte-enriched populations. Whole blood obtained from five healthy subjects was subjected to either total WBC isolation or T cell or monocyte enrichment. The cell distribution was determined by flow cytometry using labeled antibodies to the cell surface markers identified as described (16). The total WBC preparation contained predominantly neutrophils (CD66b+), but also 32% T cells and ≈8% monocytes. T cell enrichment yielded ≈95% CD2+, CD3+ cells, and monocyte enrichment yielded ≈90% CD14+, CD33+ cells.

Fig. 3.

Principal component and hierarchical cluster analyses performed on leukocyte gene expression from buffy coat and T cell- and monocyte-enriched populations. Blood was obtained from five healthy subjects, and leukocyte populations were subjected to gene expression analysis with the U133A gene chip, as described in Materials and Methods. Principal component (A) and hierarchical cluster (B) analyses were performed on the hybridization signal intensities of probe sets significant with a false discovery rate of 0.001.

Fig. 4.

Principal component and hierarchical cluster analyses performed on leukocyte gene expression from 14 trauma and 17 healthy subjects. Principal component (A) and hierarchical cluster (B) analyses were performed on the hybridization signal intensities of probe sets significant with a false discovery rate of 0.001.