Of the 72 umbilical cord samples collected for this study, 54 contained at least the minimum of 7 μg RNA required for hybridization (13 did not) and were from infants who survived to 36 weeks postmenstrual age (5 died), and therefore at risk for BPD. All expression data are available in the National Center for Biotechnology Information's Gene Expression Omnibus repository (GSE8586).
The infants included in this study were slightly more mature than those in the Extremely Low Gestational Age Newborn (ELGAN) study [13
] as a whole (Table ). This may explain why they had a higher survival rate and lower incidences of BPD and retinopathy of prematurity.
Clinical outcomes of the ELGAN (n = 1508) study population as compared with the study sample (n = 54)
Infants with and without BPD exhibited minimal differences in maternal characteristics, including cause of delivery, antenatal steroid exposure, race, or histopathologic evidence of chorioamnionitis (Table ). Infants who developed BPD were of lower birth weight and less mature gestational age, required more days of supplemental oxygen and ventilation, and had higher rates of retinopathy of prematurity. They did not differ in sex ratio or rates of patent ductus arteriosus, and had a modestly higher rate of sepsis (Table ).
Baseline characteristics of mothers and placentas related to infant outcomes in terms of BPD
Characteristics of infants in relation to the primary outcome BPD
Unsupervised learning revealed inhomogeneous clustering of the sample by gestational age (27 to 28 weeks versus < 27 weeks; data not shown) and by presence of BPD (BPD versus control; Figure ). Similarly, principal components analysis of the 54 samples across the 54,675 measured transcripts revealed partial overlap of the samples across the two groups (Figure ). Single gene differential gene expression analysis revealed no genes with a q
value (a measure of false discovery rate [FDR] [14
]) below 0.05 or an uncorrected P
value below 10-6
. The high FDR confirmed that the overlap between the two classes of infants was high; therefore, we explored systematic differences in pathways rather than individual genes, as others have done with similarly subtle differences across human samples [15
Figure 1 Unsupervised clustering based on Euclidean distance in expression between samples. Each row corresponds to a gene and each column (labeled at the bottom) corresponds to an infant who subsequently developed bronchopulmonary dysplasia (BPD) or a control (more ...)
Figure 2 Principal component analysis of the samples at birth. The closed circles correspond to infants who did not develop bronchopulmonary dysplasia (BPD) and the open circles to those who did develop BPD. The centroid of the ellipsoid hull for each subgroup (more ...)
The gene pathways most differentially expressed by gestational age category were those related to oxidative phosphorylation, mitochondrial energy metabolism, and DNA repair (Table ). None of the genes within these pathways by themselves were significantly differentially expressed, but the q
value for the entire set of genes within each pathway (of the more than 600 sets evaluated) was below 0.0001, as calculated using the sigPathway package [16
Differentially expressed gene sets for gestational age under 27 weeks versus 27 to 28 weeks
When the samples were evaluated comparing infants with BPD versus those without BPD, the most differentially expressed gene pathways included the aforementioned bioenergetic pathways, as well as histone acetyltransferase binding activity and chromatin remodeling pathways (Table ). Although the individual genes within these pathways were not significantly differentially expressed, the expression of the entire 'chromatin packaging' pathway relative to the overall transcriptome was highly significantly differentially expressed (Figure ). The ten most differentially expressed genes of those in chromatin remodeling pathways are shown in Figure to illustrate how this finding is only a trend at the individual gene level.
Differentially expressed pathways in infants with BPD versus infants without BPD
Figure 3 Relative expression of DNA packaging gene set relative to the overall transcriptome. The ratio of expression level of each gene in samples of infants that went on to develop bronchopulmonary dysplasia (BPD) against those who did not is plotted against (more ...)
Figure 4 Box-plots of differential expression of the ten most differentially expressed genes in SWI/SNF chromatin remodeling pathway. Individually, none of these genes reached significance for differential expression, although the pathway was highly significantly (more ...)