Whole Blood Gene Expression Reveals Specific Transcriptome Changes in Neonatal Encephalopathy

Abstract

Background: Variable responses to hypothermic neuroprotection are related to the clinical heterogeneity of encephalopathic babies; hence better disease stratification may facilitate the development of individualized neuroprotective therapies.

Objectives: We examined if whole blood gene expression analysis can identify specific transcriptome profiles in neonatal encephalopathy.

Material and methods: We performed next-generation sequencing on whole blood RNA from 12 babies with neonatal encephalopathy and 6 time-matched healthy term babies. Genes significantly differentially expressed between encephalopathic and control babies were identified. This set of genes was then compared to the host RNA response in septic neonates and subjected to pathway analysis.

Results: We identified 950 statistically significant genes discriminating perfectly between healthy controls and neonatal encephalopathy. The major pathways in neonatal encephalopathy were axonal guidance signaling (p = 0.0009), granulocyte adhesion and diapedesis (p = 0.003), IL-12 signaling and production in macrophages (p = 0.003), and hypoxia-inducible factor 1α signaling (p = 0.004). There were only 137 genes in common between neonatal encephalopathy and bacterial sepsis sets.

Conclusion: Babies with neonatal encephalopathy have striking differences in gene expression profiles compared with healthy control and septic babies. Gene expression profiles may be useful for disease stratification and for developing personalized neuroprotective therapies.

Keywords: Biomarkers; Brain injury; Gene expression; Neonatal encephalopathy.

Fig. 1

Two-dimensional principal component analysis plot of mean centering and scaling based on the complete gene expression. Patients are plotted according to their respective position along the two axes, either in red (neonatal encephalopathy) or blue (healthy control).

Fig. 2

Scatter plot of the top 4 significant genes according to the timing of blood sample collection (hours of age) in the neonatal encephalopathy (red) and control (blue) groups.

Fig. 3

Unsupervised hierarchical clustering of 18 babies (12 babies with neonatal encephalopathy and 6 healthy babies, horizontal axis), with 950 genes derived from negative binomial likelihood tests. Each column represents a patient and each line a gene. Upregulated genes are represented in red and downregulated genes in green. The red bar represents neonatal encephalopathy (n = 12), and the blue bar represents healthy control babies (n = 6).

Fig. 4

Canonical pathway analysis in terms of –log(p value) derived from the first 1,000 significant genes with a log₂ fold change > 1.5 in the comparison between neonatal encephalopathy and healthy control babies. The upregulated genes are shown in red and the downregulated genes are shown in green.

Fig. 5

GSE25504 microarray gene expression experiment. a Venn diagram. 4,155 genes were differentially expressed between neonatal encephalopathy and healthy controls; 684 genes were differentially expressed between bacterial sepsis and healthy controls (FDR < 0.05 and |log₂ fold change| ≥1). There were 137 genes in common between the neonatal encephalopathy and bacterial sepsis sets. b Cross-plot of the 137 shared genes (FDR < 0.05 and |log₂ fold change| ≥1) showing that only 10 genes had the same fold change direction in the two data sets.

Fig. 6

Coordination of the hypoxia response by HIF1A, MALAT1, and RICTOR. (i) In the presence of oxygen, prolyl hydroxylase (PHD) posttranslationally modifies HIF-1α, allowing it to interact with E3 ubiquitin ligase, leading to proteasomal degradation. (ii) Under hypoxic conditions, MALAT1 prevents the ubiquitination of HIF-1α, leading to HIF-1α accumulation and translocation to the nucleus, where it (iii) regulates the transcription of hypoxia-induced genes. (iv) HIF-1α mRNA translation is controlled by mTOR signaling pathways. The upregulated genes in our analysis are shown in red, while the downregulated genes are shown in green.