Functional genomic analysis of amniotic fluid cell-free mRNA suggests that oxidative stress is significant in Down syndrome fetuses

Abstract

To characterize the differences between second trimester Down syndrome (DS) and euploid fetuses, we used Affymetrix microarrays to compare gene expression in uncultured amniotic fluid supernatant samples. Functional pathway analysis highlighted the importance of oxidative stress, ion transport, and G protein signaling in the DS fetuses. Further evidence supporting these results was derived by correlating the observed gene expression patterns to those of small molecule drugs via the Connectivity Map. Our results suggest that there are secondary adverse consequences of DS evident in the second trimester, leading to testable hypotheses about possible antenatal therapy for DS.

Fig. 1.

Heatmap showing hierarchical clustering of control (C1-C7; solid red bar) and trisomic (T1-T7; solid blue bar) samples. Clustering is based on data from 409 probe sets: the 414 from the Individual gene set, minus the 5 from chromosome 21. If these five are not removed, the dendrogram looks quite similar; in particular, it similarly separates the control and trisomic samples. Expression values in each row are z-score normalized.

Fig. 2.

Histogram of fold-changes in gene expression between the average expression in DS and the average in the controls. (A) Histogram for all 501 probe sets representing genes on chromosome 21. The fold-changes are reported in log-scale (base 2), so that up- and down-regulation appear at equal distances from zero. For example, a value of 1.0 represents 2-fold up-regulation in DS. The bold vertical line at ≈0.58 corresponds to the 1.5-fold change expected from the gene-dosage hypothesis. (B) Histogram of all probe sets representing genes on chromosomes other than 21, shown on the same scale as in A).

Fig. 3.

Putative network of pathways implicated in DS. Significantly implicated processes, based on DAVID functional analysis, are shown in boxes. Edges between boxes represent relationships between functional processes such as G protein signaling and disruptions in ion transport that result from oxidative stress, as suggested in refs. (solid lines), (dotted lines), (dashed lines), and (dash-dotted lines). Blue boxes with dashed borders are processes over-represented in the Individual gene set only; magenta boxes with thin solid borders are processes over-represented in both the Individual and Leading Edge gene sets; and green boxes with thick solid borders are implicated by both the Individual and Leading Edge gene sets and by the Connectivity Map.