Candidate biomarkers and persistent transcriptional responses after low and high dose ionizing radiation at high dose rate

Figures & data

Table 1. Microarray datasets studied.

	GSE8917	GSE43151	GSE23515
Study & Year	Paul & Amundson, 2008	Nosel et al., 2013	Paul & Amundson, 2011
Platform	Agilent Whole Human Genome Oligo Microarrays (D4112A)	Agilent Whole Human Genome Microarray v2, 4x44K	Agilent Whole Human Genome Oligo Microarrays (D4112A)
Donors	10 healthy donors (5 females/ 5 males)	5 healthy donors (5 males)	24 healthy donors (12 females/ 12 males)
Samples	Whole blood in sodium citrate	Whole blood in heparin diluted 1:10 in IMDM	Whole blood in sodium citrate
RNA Extraction Sources	Whole blood diluted in RPMI-1640/ 10% FBS	CD4+ T lymphocytes	Whole blood diluted in RPMI-1640/ 10% FBS
Dose (Gy)	0.5, 2, 5, 8	0.005, 0.01, 0.025, 0.05, 0.1, 0.5	0.1, 0.5, 2
Dose Rate (Gy/min)	0.82	0.05	0.82
Time after IR (hours)	6, 24	2.5, 5, 7.5, 10	6

IMDM: Iscove’s Modified dulbecco’s Medium; RPMI: Roswell park memorial institute; FBS: fetal bovineserum.

Figure 1. Differentially expressed (DE) genes after low- and high-dose IR exposures. A: Venn diagram of the DE genes. B: the percentages of identified DE genes that Overlapped with known functional databases. C: top 5 bioprocesses (biological process), KEGG (Kyoto Encyclopdia of genes and Genomes), and Wiki (Wikipedia) pathways enriched in DE genes after low-dose IR exposure. D: top 5 biological process, KEGG and Wiki pathways enriched in DE genes after high-dose IR exposure.

Figure 2. DE genes with known cell-cycle phases and checkpoint functions, where genes in red are upregulated with a positive ${\beta }_1,$ and genes in blue are down-Regulated with a negative ${\beta }_1.$ A: Cell cycle related DE genes after high-dose IR exposure (GSE8917). B: Cell cycle associated DE genes after low-dose IR exposure (GSE43151).

Figure 3. Average log-fold changes (LFC) of the DE genes in each cluster over time and dose DE genes are divided into 4 clusters with the sign of ${\beta }_1$ and ${\beta }_2,$ where cluster I (C1) contains DE genes with ${\beta }_1$ and ${\beta }_2$ oth being positive (+ +); cluster 2 (C2) consists of DE genes with positive ${\beta }_1$ but negative ${\beta }_2$ (+ -); cluster 3 (C3) comprises DE genes with negative ${\beta }_1$ but positive ${\beta }_2$ (- +); and cluster 4 (C4) includes DE genes with ${\beta }_1$ and ${\beta }_2$ both being negative (- -). A: the 4 clusters after high-dose (HD) IR exposure. B: the 4 clusters after low-dose (LD) IR exposure. From left to right, columns 1 and 3 are the 3D plots for LFC vs both dose and time course, while columns 2 and 4 are the marginal 2D plots for LFC against dose and time, separately.

Figure 4. Pie charts showing the percentage of DE genes in different clusters and the KEGG pathway enrichment analysis for genes in clusters C1 (${\beta }_1$ > 0 & ${\beta }_2$ > 0) and C4 (${\beta }_1$ < 0 & ${\beta }_2$ < 0) following low- and high-dose IR exposures. LD-C1: Top 5 KEGG (Kyoto Encyclopedia of genes and Genomes) pathways for upregulated genes dose IR exposure; and HD-C1: Top 5 KEGG pathways for upregulated genes in C1 after a high-dose IR exposure. There is no KEGG pathway enriched for downregulated genes in LD-C4.

Table 2. Common DE genes over high- and low-dose IR exposure.

	High-Dose Irradiation (GSE8917)				Low-Dose Irradiation (GSE43151)
Gene ID	${\mathit{\beta }}_1$	${\mathit{\beta }}_2$	${\mathit{P}}_2$	${\mathit{C}}_{\mathit{H}}$	${\mathit{\beta }}_1$	${\mathit{\beta }}_2$	${\mathit{P}}_2$	${\mathit{C}}_{\mathit{L}}$
ARHGEF3	0.449	−0.428	1.72E-06	C2	0.512	−0.089	2.46E-01	C2
BAX	0.317	0.062	4.60E-01	C1	1.287	0.282	3.40E-02	C1
BBC3	0.672	−0.026	8.18E-01	C2	2.187	−0.547	2.55E-03	C2
CCDC109B	−0.644	0.230	3.56E-03	C3	−0.254	0.105	6.18E-02	C3
DCP1B	0.613	−0.670	2.15E-09	C2	0.648	−0.091	1.15E-01	C2
DDB2	1.308	−0.531	2.43E-04	C2	1.394	−0.128	2.44E-01	C2
F11R	0.586	0.211	3.22E-02	C1	0.175	0.138	4.39E-04	C1
GADD45A	1.399	−0.000	9.99E-01	C2	1.086	−0.703	3.25E-11	C2
GSS	0.428	0.060	3.57E-01	C1	0.260	0.240	1.61E-04	C1
PLK3	0.688	−0.079	3.06E-01	C2	0.453	−0.819	3.96E-20	C2
TNFRSF10B	1.085	0.095	5.30E-01	C1	1.444	0.510	6.33E-08	C1
XPC	0.964	−0.360	3.30E-03	C2	0.901	0.042	5.68E-01	C1
CBX3	−0.348	0.054	3.52E-01	C3	0.279	−0.040	5.26E-01	C2
PPP3CC	−0.427	−0.127	1.03E-01	C4	0.176	0.451	2.01E-16	C1
RNF113A	−0.359	−0.139	2.19E-02	C4	0.201	−0.032	4.68E-01	C2
ASCC3	1.176	−0.452	1.22E-02	C2	1.093	0.811	7.58E-10	C1
FBXO22	0.864	−0.542	9.87E-04	C2	0.522	0.253	1.19E-06	C1
FBXW7	0.455	−0.447	5.40E-06	C2	0.306	0.686	1.87E-18	C1
FDXR	1.971	−2.028	1.01E-12	C2	2.826	1.668	3.71E-14	C1
PCNA	1.744	−0.749	2.81E-05	C2	1.338	0.636	5.13E-10	C1
PHPT1	0.908	−1.203	8.07E-14	C2	1.634	0.794	3.89E-08	C1
PPM1D	0.893	−0.340	1.06E-02	C2	0.471	0.187	2.94E-04	C1
REV3L	0.428	−0.254	9.34E-04	C2	0.350	0.302	2.78E-08	C1
SESN1	0.799	−0.650	2.64E-07	C2	0.615	0.377	2.21E-04	C1
SRA1	0.582	0.266	1.37E-02	C1	0.298	−0.204	4.70E-03	C2

The DE genes were selected with $P_1<1e-05$ (not reported in the Table). We report the estimated coefficients ${\mathrm{\beta }}_1$ and ${\beta }_2$ for dose and time, the corresponding P value ($P_2)$ of ${\beta }_2,$ and the clusters each gene belongs to. Genes in yellow are the potential transcriptional markers for dosimetry. The middle 3 genes in orange have opposite dose effects after low- and high-dose exposures. The unshaded genes at the bottom have opposite time effects after low- and high-dose irradiations. The full description of the identified genes can be found at https://www.genecards.org/.

Figure 5. Boxplots of the LFC of 10 candidate biomarkers following different doses of IR exposure (0.1 Gy, 0.5 Gy, and 2 Gy) in the validation dataset (GSE23515). the y-Axis represents LFC. The red dot and the horizontal line inside each box represent the median.

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