Supplementary Material for: Differential Gene Expression in Cord Blood of Infants Diagnosed with Cerebral Palsy: a Pilot Analysis of the BEAM Cohort

The Beneficial Effects of Antenatal Magnesium (BEAM) clinical trial was conducted between 1997-2007, and demonstrated a significant reduction in cerebral palsy (CP) in preterm infants who were exposed to peripartum magnesium sulfate (MgSO4). However, the mechanism by which MgSO4 confers neuroprotection remains incompletely understood. Cord blood samples from this study were interrogated during an era when next generation sequencing was not widely accessible and few gene expression differences or biomarkers were identified between treatment groups. Our goal was to use bulk RNA deep sequencing to identify differentially expressed genes comparing the following four groups: newborns who ultimately developed CP treated with MgSO4 or placebo, and controls (newborns who ultimately did not develop CP) treated with MgSO4 or placebo. Those who died after birth were excluded. We found that MgSO4 upregulated expression of SCN5A only in the control group, with no change in gene expression in cord blood of newborns who ultimately developed CP. Regardless of MgSO4 exposure, expression of NPBWR1 and FTO were upregulated in cord blood of newborns who ultimately developed CP compared with controls. These data support that MgSO4 may not exert its neuroprotective effect through changes in gene expression. Moreover, NPBWR1 and FTO may be useful as biomarkers, and may suggest new mechanistic pathways to pursue in understanding the pathogenesis of CP. The small number of cases ultimately available for this secondary analysis, with male predominance and mild CP phenotype, is a limitation of the study. In addition, differentially expressed genes were not validated by qRT-PCR.