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.

本项关于产前镁剂（BEAM）的有益效应的临床试验于1997年至2007年间进行，并证实了在暴露于围产期硫酸镁（MgSO4）的早产儿中，脑瘫（CP）的发生率显著降低。然而，MgSO4提供神经保护作用的机制尚不完全明了。在本研究中，当下一代测序技术尚未广泛应用且治疗组之间识别出的基因表达差异或生物标志物寥寥无几的时期，对研究中的脐带血样本进行了分析。我们的目标是利用大批量RNA深度测序技术，比较以下四组新生儿的差异表达基因：最终发展为CP并接受MgSO4或安慰剂治疗的新生儿，以及未发展为CP的对照组（接受MgSO4或安慰剂治疗的新生儿）。出生后死亡的婴儿被排除在外。我们发现，仅在对照组中，MgSO4上调了SCN5A的表达，而在最终发展为CP的新生儿脐带血中未观察到基因表达的变化。无论是否暴露于MgSO4，与对照组相比，最终发展为CP的新生儿脐带血中NPBWR1和FTO的表达均上调。这些数据支持了MgSO4可能并非通过改变基因表达来发挥其神经保护作用的观点。此外，NPBWR1和FTO可能作为生物标志物具有潜在价值，并可能为探索CP发病机制的新的作用途径提供线索。然而，本次二次分析中可供分析的病例数量有限，以男性为主，且CP表型轻微，这构成了研究的局限性。此外，差异表达基因尚未通过qRT-PCR进行验证。