3 Statistical analysis of microarray experiments
4.3 Results
4.3.2 Differential expression analysis of BPD severity groups using LIMMA
Supervised clustering reveals distinct gene expression pattern discriminating preterm infants with lower and higher BPD grades at birth.
The supervised approach of the microarray analysis revealed 238 differentially regulated transcripts with a FDR < 0.05, and |FC| > 2 out of 7529 transcripts (see Figure 4-2).
Hierarchical clustering of samples with these differentially regulated genes reveals two main clusters of expression profiles. One cluster contains all microarray patterns of preterm infants with mild BPD. The other cluster can be divided into a subcluster of no BPD preterm infants and a subcluster, which contains all samples of preterm infant of group 3. The expression patterns indicate that processes at birth in the group of no BPD and moderate to severe BPD are more similar to each other than to the group of patients, who developed a mild BPD.
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Figure 4-1 Total of 238 differentially expressed genes in comparison of three BPD severity grades (no, mild, and moderate-severe BPD)
Most of the differentially expressed transcripts can be found in the comparison of preterm infants without BPD and with mild BPD (Figure 4-1). In this comparison, 127 transcripts are differentially expressed, 84 are up regulated, and 43 are down regulated in infants with mild BPD compared to preterm infants without BPD. Preterm infants with mild BPD also show a distinct pattern compared to preterm infants with moderate or severe BPD with 125 (62 up, 63 down regulated) significantly differentially regulated transcripts.
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Figure 4-2 Expression profiles of significantly differentially regulated genes in preterm infants reveal two main hierarchical clusters of expression.
Preterm infants with BPD grade 1 are depicted in blue, with BPD grade 2 or 3 in green, and infants without BPD in red in the upper hierarchical clustering dendrogram. Hierarchical clustering was performed on scaled data with Euclidean distance measure and Ward’s Linkage clustering method.
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A high number of differentially regulated genes in the comparisons of group II vs. I and group II vs. III of 39 transcripts indicates that the gene expression pattern in preterm infants developing mild BPD differs greatly from no BPD and moderate to severe BPD gene expression. The development of mild BPD follows different routes than the development of more severe BPD. The most down regulated transcripts in mild BPD are CRIP1 (Cysteine-rich protein 1 (intestinal)), NM_005129, PTPRCAP (Protein tyrosine phosphatase, receptor type, C-associated protein); the most up regulated transcripts in mild BPD are GAL (Galanin prepropeptide), SLC24A3 (Solute carrier family 24 (sodium/potassium/calcium exchanger), member 3), and CDH13 (Cadherin 13, H-cadherin (heart)) (see Table 8-1 in
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Appendix). DAVID functional annotation clustering indicates an overrepresentation of these genes associated with the cytoskeleton and calcium homeostasis. Transcripts which are only significantly differentially regulated in group II (mild BPD) compared to group I (no BPD) preterm infants are putatively overrepresented in the membrane/glycoprotein, transmembrane transport, and leukocyte activation. Transcripts only differentially regulated between group II and group III (moderate/severe BPD) are putatively overrepresented in the mitochondrion or oxidation reduction, and regulation of cell migration.
Group III preterm infants express 27 genes differentially, when compared to preterm infants who develop mild or no BPD (see Table 8-2 in
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Appendix). The most up regulated transcripts at birth in preterm infants at birth with moderate/severe BPD are HPR (Haptoglobin-related protein), MAP4K3 (Mitogen-activated protein kinase 3), and PGLYRP1 (Peptidoglycan recognition protein 1), CDA (Cytidine deaminase). The most down regulated genes in moderate or severe BPD are FKBP14 (FK506 binding protein 14), GNG11 (Guanine nucleotide binding protein (G protein), gamma 11), and ETNK1 (Ethanolamine kinase 1). DAVID functional annotation clustering indicates an overrepresentation of genes associated with transcription or transcription factor activity.
Group III preterm infants differ in the expression of 48 transcripts (32 up regulated, 16 down regulated) from infants without BPD. A total of 4 transcripts, i.e. ACTN2 (Actinin, alpha 2), NM_003832, NM_018104, and SLC2A11 (Solute carrier family 2 (facilitated glucose transporter), member 11), is able to differentiate between mild BPD and no BPD preterm infants, and is also differentially regulated in preterm infants with moderate/severe BPD and infants without BPD (see Table 8-3 in
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Appendix). Transcripts only significantly differentially regulated in group III preterm infants compared to no BPD infants are putatively overrepresented in the biological processes:
induction of apoptosis, regulation of transcription. For the transcripts differentially regulated in both group II and group III preterm infants compared to no BPD infants, no functional clustering analysis can be conducted.
Principal component analysis reveals a clear distinction between preterm infants without BPD, mild BPD, and moderate/severe BPD (Figure 4-3). In hierarchical cluster analysis it can be seen that the gene expression pattern of preterm infants without BPD is more similar to gene expression in preterm infants with moderate or severe BPD than with mild BPD.
Figure 4-3 Principal components analysis using 238 differentially regulated genes identified by gene expression analysis of BPD severity groups
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At time of birth differentially regulated genes in preterm infants are involved in chemotaxis and leukocytes.
An IPA comparison analysis for the unsupervised analysis approach predicted (1) activated chemotaxis of cells and (2) decreased biological functions associated with apoptosis and accumulation of leukocytes in preterm infants developing mild BPD (vs. no and moderate/severe BPD) as well as (3) decreased phagocytosis in infants with moderate/severe BPD (vs. no BPD) (see Table 4-3).
Table 4-3 Biological functions predicted to be increased or decreased at time of birth in preterm infants developing BPD
Shown are functions with z-scores ≥ |1.5|, which indicate the direction of activation; processes with positive z-scores are predicted to increased, negative z-scores indicate a decreased function;
asterisks indicate significance level of enrichment: * p < 0.05, ** p < 0.01, * p < 0.001.
Diseases and Bio Functions mild vs. no
BPD
mod./s.
vs. no BPD
mod./s.
vs. mild BPD
accumulation of cells -2.42 *
apoptosis of cancer cells -2.23 *
damage of kidney -2.22 *
accumulation of granulocytes -2.22 **
cell death of cancer cells -2.22 *
accumulation of leukocytes -2.20 **
accumulation of eosinophils -1.98 ***
weight loss -1.71 *
phagocytosis -1.73 *
branching of neurites
1.96 *
cell movement of vascular smooth muscle cells
-2.00 *
chemotaxis of cells 2.66 *
-1.86 *
aggregation of cells 1.95 ** -0.73 *
chemotaxis of leukocytes 2.28 *
quantity of granulocytes 2.18 *
chemotaxis of mononuclear leukocytes 2.11 *
quantity of neutrophils 2.06 *
airway hyperresponsiveness 1.85 **
chemotaxis of lymphocytes 1.83 *
86 Cytokines as upstream regulators in mild BPD
Cytokines play an important role in the innate immunity of preterm infants (Melville and Moss, 2013). Therefore the upstream regulator analysis (see chapter 3.4.3 functional gene annotation analysis with IPA) is focused on cytokines with the aim to identify potential cytokines as biomarkers present in the cord blood of preterm infants (see Table 4-4).
Table 4-4 Cytokine upstream regulators (IPA) predicted to be activated or deactivated at birth of preterm infants developing BPD
Upstream regulators mild vs. no BPD
mod./s. vs. no BPD
mod./s. vs.
mild BPD
IL2 1.80
TNF 1.66 **
IL6 1.52 * 0.00 *
IL10 1.21 *
IFN alpha/beta 1.13 **
IFNG 0.56 *
CCL19 0.00 **
CCL8 0.00 *
CXCL9 0.00 *
Ifn gamma 0.00 **
IFNE 0.00 **
IFNK 0.00 *
IFNW1 0.00 **
Mac 0.00 *
TSLP 0.00 *
CSF3 0.00 *
EBI3 0.00 **
IFN Beta 0.00 *
IFNA1/IFNA13 0.00 *
IFNA10 0.00 *
IFNA14 0.00 *
IFNA17 0.00 *
IFNA21 0.00 *
IFNA4 0.00 *
IFNA5 0.00 *
IFNA6 0.00 *
IFNA7 0.00 *
IFNA8 0.00 *
IL27 0.00 **
IL5 2.00
IL8 0.00 *
WNT1 0.00 *
Shown are z-scores, which indicate the direction of activation; regulators with positive z-scores are predicted to activated (red); asterisks indicate significance level for enrichment: * p < 0.05, ** p < 0.01, * p < 0.001.
In mild BPD compared to no BPD TNF- and interleukins (IL-2, IL-6, and IL-10) are the highest activated cytokines, but also interferons and chemokines are predicted to be
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activated. Differentially regulated genes in moderate/severe BPD infants on the other hand are mainly regulated by interferons.
Networks combining differentially expressed genes, upstream regulators, and downstream effects, so called regulator effects networks, of differentially regulated genes in preterm infants with mild BPD compared to infants with no BPD firstly demonstrated a relationship between the activation of IL-6, TCR (T cell receptor), TNF- and the regulation of CXCL9 (chemokine ligand 9), IL-10, LAT (Linker for activation of T cells), LGALS3 (lectin, galactoside-binding, soluble, 3), MMP7 (matrix metallopeptidase 7), TLR3 (toll-like receptor 3), TNFRSF1A (tumor necrosis factor receptor superfamily, member 1a) (Figure 4-4). The regulation of these genes is linked to a predicted activation of the function chemotaxis of leukocytes, and the deactivation of the accumulation of eosinophils and fibrosis.
Figure 4-4 Regulator effector networks linking TCR, TNF-α, IL-6 activation to activation of chemotaxis of leukocytes and deactivation of fibrosis and accumulation of eosinophils in preterm infants with mild BPD compared to preterm infants without BPD
Secondly, a relationship between the predicted activation of MAPK14 (mitogen-activated protein kinase 14), the differential expression CAT (catalase), IL-10, SREBF1 (sterol
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regulatory element binding transcription factor 1), ZFP36 (zinc finger protein 36), and the predicted deactivation of disease functions leading to apoptosis and necrosis (damage of kidney, cell death of cancer cells) was found (Figure 4-5).
Figure 4-5 Regulator effector networks linking MAPK14 activation and the inhibition of functions leading to apoptosis and necrosis in preterm infants with mild BPD compared to preterm infants without BPD
The activation of TNF- was furthermore predicted by the expression of CAT, CDH13 (cadherin 13), IL-10, LGALS3, TNFRSF1A which in turn had been linked to airway hyperresponsiveness in preterm infants with mild BPD (Figure 4-6).
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Figure 4-6 Regulator effector networks linking TNF-α activation to the activation of airway hyperresponsiveness in preterm infants with mild BPD compared to preterm infants without BPD
Figure 4-7 Regulator effector networks linking TNF-α activation to the activation of proliferation of granulocytes in preterm infants with mild BPD compared to preterm infants without BPD
An additional prediction analysis showed the activation of TNF- by BID (BH3 interacting domain death agonist), CDH13, IL-10, LGALS3, TF (transferrin), TNFRSF1A, ZFP36, involved in granulocytes proliferation (Figure 4-7).
Increased neutrophil number is predicted as a consequence of up-regulated IL-10, TNF-, TNFRSF1A through the regulator IL-6 (Figure 4-8).
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Figure 4-8 Regulator effector networks linking IL-6 activation to the activation of proliferation of neutrophils in preterm infants with mild BPD compared to preterm infants without BPD
4.3.3 Predictive Microarray Analysis (PAM) for preterm infants with and