Alcohol Use During Pregnancy is Associated with Specific Alterations in MicroRNA Levels in Maternal Serum

Despite evidence that consuming alcohol during pregnancy is harmful for the developing fetus, more than 10% of women in the United States report at least some alcohol use during pregnancy (Tan et al., 2015). The prevalence of fetal alcohol syndrome in the United States ranges from 0.5 to 2.0 per 1,000 live births in the general population to 1.4 to 9.8 per 1,000 live births in high‐risk groups (May and Gossage, 2001). Based on active case ascertainment methods, the prevalence of the entire continuum of fetal alcohol spectrum disorder (FASD) has been estimated to be as high as 24 to 48 per 1,000 live births, or 2 to 5% in the United States (May et al., 2014). However, the identification of children adversely affected by prenatal alcohol exposure (PAE) can be difficult because many of them do not exhibit any of the physical features associated with PAE and mainly manifest with neurocognitive deficits later in life. In the absence of the physical features and without accurate information on PAE, it is difficult to make a diagnosis of FASD. While ethanol (EtOH) biomarkers, as an objective measure, can help to overcome the limitations of self‐report on alcohol use during pregnancy, none of the existing biomarkers of alcohol consumption are 100% sensitive or specific (Bakhireva and Savage, 2011). Given these limitations, the identification of more sensitive and specific biomarkers that either alone or in combination with other clinical and self‐report measures can provide more accurate information on PAE would result in better identification of children with FASD and increase the opportunities for earlier diagnosis and interventions. MicroRNAs (miRNAs) are approximately 22‐nucleotide single‐stranded non‐protein‐coding RNA molecules that serve primarily to silence the expression of mRNA transcripts to which they hybridize by base pairing. miRNA biogenesis involves the processing of primary miRNA transcripts (pri‐miRNA) to hairpin precursor structures (pre‐miRNA), which are then exported to the cytoplasm where either strand may be loaded into the RNA‐induced silencing complex (RISC). The strand previously called the miRNA star (miRNA*) was generally thought to be rapidly degraded. However, miRNA* sequences have been found in RISC and shown to impact disease states such as cancer (Jazdzewski et al., 2009). The nomenclature is now changing to describe from which arm of the precursor the miRNA originates, 5′ (5p) or 3′ (3p). Mature miRNAs can bind thousands of transcripts in a cell, making them “master regulators” of gene expression (Bartel, 2004). Moreover, miRNAs have been shown to control many complex biological processes, from embryonic development to the promotion (or inhibition) of various pathological conditions, including cancers and neurodevelopmental disorders (Sayed and Abdellatif, 2011). Recent animal model and cell culture studies suggest that miRNAs play an important role in the mechanisms underlying the deleterious effects of PAE (Balaraman et al., 2013). EtOH suppresses miRs‐9, ‐21, ‐153, and ‐335 in cultured fetal mouse neural stem cells, coordinately regulating genes that make cells resistant to apoptosis, with increased proliferation and aberrant differentiation (Sathyan et al., 2007). miRs‐9(‐5p), 9*(‐3p), and ‐153 are repressed by EtOH exposure in the developing zebrafish brain (Tal et al., 2012). EtOH exposure in the developing mouse brain increases the expression of miRs‐10a and ‐10b, with a concomitant decrease in the target gene homeobox A1 (HOXA1; Wang et al., 2009), which is essential for normal embryonic development. Alterations in miRNA expression were also found in primary neuronal cultures from the cortex of mice at embryonic day 15 following chronic intermittent EtOH exposure and withdrawal (Guo et al., 2012). Finally, differences in miRNA expression were seen in the amygdala and striatum of rats at postnatal day 42 that were exposed to EtOH prenatally and subjected to social enrichment training postnatally (Ignacio et al., 2014). In addition to working inside the cell, miRNAs can be secreted and transferred between cells, indicating that they participate in cell‐to‐cell communication (Valadi et al., 2007). miRNAs are present not only in blood plasma and serum but also in a variety of cell‐free body fluids including urine and saliva, making them attractive as potential biomarkers. In the circulation, they are found in vesicles such as exosomes, microvesicles, and apoptotic bodies (Valadi et al., 2007; Zernecke et al., 2009) as well as in association with proteins (Vickers et al., 2011). Because of their association with vesicles and proteins, circulating miRNAs are resistant to degradation and can be accurately measured even when present at very low levels (Mitchell et al., 2008). Thus, circulating miRNAs have great promise as biomarkers of a variety of disease conditions from cancer to myocardial infarction (Etheridge et al., 2011). Although extracellular miRNAs have been investigated in animal models of PAE (Balaraman et al., 2014), the utility of these biomarkers in humans has not been reported thus far. The goal of the present study was to identify alterations in miRNA expression in maternal serum that could predict maternal alcohol consumption in pregnant women and further elucidate the mechanisms of fetal alcohol effects.