Gene expression signature for early prediction of late occurring pancytopenia in irradiated baboons

Abstract

Based on gene expression changes measured in the peripheral blood within the first 2 days after irradiation, we predicted a pancytopenia in a baboon model. Eighteen baboons were irradiated with 2.5 or 5 Gy. According to changes in blood cell counts, the surviving baboons (n = 17) exhibited a hematological acute radiation syndrome (HARS) either with or without a pancytopenia. We used a two stage study design where stage I was a whole genome screen (microarrays) for mRNA combined with a qRT-PCR platform for simultaneous detection of 667 miRNAs using a part of the samples. Candidate mRNAs and miRNAs differentially upregulated or downregulated (>2-fold, p < 0.05) during the first 2 days after irradiation were chosen for validation in stage II using the remaining samples and using throughout more sensitive qRT-PCR. We detected about twice as many upregulated (mean 2128) than downregulated genes (mean 789) in baboons developing an HARS either with or without a pancytopenia. From 51 candidate mRNAs altogether, 11 mRNAs were validated using qRT-PCR. These mRNAs showed only significant differences between HARS groups and H0, but not between HARS groups with and without pancytopenia. Six miRNA species (e.g., miR-574-3p, p = 0.009, ROC = 0.94) revealed significant gene expression differences between HARS groups with and without pancytopenia and are known to sensitize irradiated cells. Hence, in particular, the newly identified miRNA species for prediction of pancytopenia will support the medical management decision making.

Keywords: Gene expression; Hematological acute radiation syndrome (HARS); Pancytopenia; miRNA.

Fig. 1

Changes in blood cell counts of neutrophils (upper graph), platelets (middle graph), and red blood cells (hemoglobin, lower graph) are shown for all 18 baboons up to 203 days after exposure. HARS severity was determined separately for count changes in neutrophils, lymphocytes, and platelets during the whole follow-up starting at day 7. Gray dashed lines indicate limits (neutrophils: 0.5 × 1000/μl; platelets, 10 × 1000/μl; red blood cells/hemoglobin, 8 g/dl) for the definition of a pancytopenia

Fig. 2

Venn diagrams showing the number of upregulated (left side) and downregulated (right side) protein coding genes (mRNA transcripts) observed for HARS with pancytopenia and HARS without pancytopenia. Differentially expressed genes (DEG) observed on both days after exposure are shown in the overlapping circle. Numbers outside the overlapping region represent the total number of differentially expressed genes that were not in common over day 1 to day 2. Percentages in parenthesis refer to the number of overlapping genes relative to the DEG of day 1 (first entry in parenthesis) and day 2 (second entry in parenthesis)

Fig. 3

Gene expression changes in the peripheral blood were examined in baboon groups being either unexposed (H0) or belonging to HARS groups with and without pancytopenia. We plotted the fold change (FC) of candidate genes from the screening stage relative to the mean H0 values on day 1 (miR-29c and miR-574-3p) and day 2 (miR-133a) after exposure. FCs of the genes from the screening stage are shown at the left side, and corresponding data from the validation stage are shown on the right side of the graphs. The validation stage includes additional samples not used during the screening stage I (see also Table 1). Symbols represent mean values and error bars reflect the SEM. The number of measurements per group is shown in Table 3