Analysis of MicroRNA Expression in Newborns with Differential Birth Weight Using Newborn Screening Cards

Abstract

Birth weight is an early predictor for metabolic diseases and microRNAs (miRNAs) are proposed as fetal programming participants. To evaluate the use of dried blood spots (DBS) on newborn screening cards (NSC) as a source of analyzable miRNAs, we optimized a commercial protocol to recover total miRNA from normal birth weight (NBW, n = 17-20), low birth weight (LBW, n = 17-20) and high birth weight (macrosomia, n = 17-20) newborns and analyzed the relative expression of selected miRNAs by stem-loop RT-qPCR. The possible role of miRNAs on the fetal programming of metabolic diseases was explored by bioinformatic tools. The optimized extraction of RNA resulted in a 1.2-fold enrichment of miRNAs respect to the commercial kit. miR-33b and miR-375 were overexpressed in macrosomia 9.8-fold (p < 0.001) and 1.7-fold, (p < 0.05), respectively and miR-454-3p was overexpressed in both LBW and macrosomia (19.7-fold, p < 0.001 and 10.8-fold, p < 0.001, respectively), as compared to NBW. Potential target genes for these miRNAs are associated to cyclic-guanosine monophosphate (cGMP)-dependent protein kinase (PKG), mitogen-activated protein kinase (MAPK), type 2 diabetes, transforming growth factor-β (TGF-β)and Forkhead box O protein (FoxO) pathways. In summary, we improved a protocol for analyzing miRNAs from NSC and provide the first evidence that birth weight modifies the expression of miRNAs associated to adult metabolic dysfunctions. Our work suggests archived NSC are an invaluable resource in the search for fetal programming biomarkers.

Keywords: birth weight; circulating microRNAs; fetal programming; newborn screening cards.

Figure 1

Extraction protocols and representative miRNA yield from neonatal screening cards (NSC). (A) Flow diagram of 4 out of 11 extraction protocols performed. (B) Gel-like image for small RNA quality from 11 protocols for miRNA purification performed from dried blood samples on NSC. (C) Electropherogram from protocols 3 (purple), 4 (orange), 7 (magenta) and 8 (gold). (D) miRNA:small RNA ratio from NSC of normal (green), low body weight (red) and macrosomia (blue) neonates, horizontal line indicates the 0.35 threshold ratio, each bar represents an individual, n = 20. NBW, normal birth weight; LBM, low birth weight.

Figure 2

Normalized miRNA expression relative to normal birth weight. Newborn expression (log2 transformed) of miR-33b (A), miR-375 (B) and miR-454-3p (C) from normal birth weight (green), low birth weight (red) and macrosomia (blue) neonates. Bars represent the mean ± SEM. * (p < 0.05) and *** (p < 0.001) respect to normal birth weight, by Tukey’s honest significant difference (HSD) test. n = 17–20.

Figure 3

Venn diagrams illustrating the bioinformatics analysis of target genes for miR-33b (A), miR-375 (B) and miR-454-3p (C). For each miRNA, at least four public online database algorithms were used to identify target mRNAs. The overlap shows the number of targets shared by the algorithms, increasing the number of candidate genes with strong likelihood to be experimentally validated.