Neuropeptide Y2 Receptor (NPY2R) Expression in Saliva Predicts Feeding Immaturity in the Premature Neonate

Abstract

BackgroundThe current practice in newborn medicine is to subjectively assess when a premature infant is ready to feed by mouth. When the assessment is inaccurate, the resulting feeding morbidities may be significant, resulting in long-term health consequences and millions of health care dollars annually. We hypothesized that the developmental maturation of hypothalamic regulation of feeding behavior is a predictor of successful oral feeding in the premature infant. To test this hypothesis, we analyzed the gene expression of neuropeptide Y2 receptor (NPY2R), a known hypothalamic regulator of feeding behavior, in neonatal saliva to determine its role as a biomarker in predicting oral feeding success in the neonate.Methodology/Principal FindingsSalivary samples (n = 116), were prospectively collected from 63 preterm and 13 term neonates (post-conceptual age (PCA) 26 4/7 to 41 4/7 weeks) from five predefined feeding stages. Expression of NPY2R in neonatal saliva was determined by multiplex RT-qPCR amplification. Expression results were retrospectively correlated with feeding status at time of sample collection. Statistical analysis revealed that expression of NPY2R had a 95% positive predictive value for feeding immaturity. NPY2R expression statistically significantly decreased with advancing PCA (Wilcoxon test p value<0.01), and was associated with feeding status (chi square p value = 0.013).Conclusions/SignificanceDevelopmental maturation of hypothalamic regulation of feeding behavior is an essential component of oral feeding success in the newborn. NPY2R expression in neonatal saliva is predictive of an immature feeding pattern. It is a clinically relevant biomarker that may be monitored in saliva to improve clinical care and reduce significant feeding-associated morbidities that affect the premature neonate.

Comparative performance analyses of commercially available products for salivary collection and nucleic acid processing in the newborn

“…We have shown earlier that GAPDH is a stable reference gene among premature neonates (Maron et al 2012). Owing to limited sample volume, each sample was run only once.…”

The Neonatal Salivary Transcriptome

“…More recently, Pandit and colleagues (2013) have modified an existing Qiagen protocol (QIAzol), to provide investigators with a robust, cost-effective alternative to other commercially available RNA extraction kits. By comparison, published reports in the newborn have successfully used Qiagen's RNAProtect Saliva Solution on multiple downstream transcriptomic platforms (Maron et al , 2012bDietz et al 2012). Thus, investigators have options when considering salivary collection and extraction kits and should consider study design, location and costs when choosing a product or protocol.…”

“…Most salivary collection kits recommend 1 mL of saliva for downstream RNA extraction and analysis. However, neonates on average can only generate 50 mL of saliva per sample (Maron et al , 2012b. Despite these small volumes, successful extraction and identification of RNA targets has been performed repeatedly in the newborn.…”

“…However, a series of published reports have confirmed the presence of saliva mRNAs, leaving little doubt of their potential for enhancing and informing clinical care (Li et al 2004a;Hu et al 2006Hu et al , 2008Park et al 2007;Maron et al 2010;Lee et al 2011). Extracted mRNAs have been applied to multiple downstream transcriptomic platforms including microarrays (Li et al 2004a), RT-qPCR (Maron et al 2012b), and RNA sequencing (Spielmann et al 2012). Each technique offers the investigator a slightly different approach to interrogate the transcriptome.…”

“…Because each study subject learned to orally feed during his/her hospitalization, genes identified in the initial study mapped to biological processes involved in cranial nerve development, facial morphogenesis, neurodevelopment, and feeding behavior (Maron 2011). Within the feeding behavior category, salivary gene expression of the neuropeptide Y2 receptor (NPY2R) was subsequently shown to be a highly informative biomarker of feeding immaturity in this population (Maron et al 2012b).…”