Jill L. Maron

Prenatal diagnosis using cell‐free nucleic acids in maternal body fluids: A decade of progress

The ability to detect cell‐free fetal nucleic acids in pregnant women has greatly evolved over the past decade. Dozens of papers have explored the biology, kinetics, and clinical significance of both cell‐free fetal DNA and mRNA in the maternal circulation. As a result, our overall understanding of fetal nucleic acid trafficking has expanded. To date, two applications, gender determination and fetal RhD status, have translated into clinical medicine. However, with advanced molecular techniques such as mass spectrometry, real‐time quantitative polymerase chain reaction, and gene expression arrays, the ease with which fetal genes can be detected within the mother has greatly improved. Newly identified placental and fetal mRNA transcripts as well as an epigenetically modified placental DNA marker, maspin, have universal applicability. Global expression analyses of fetal mRNA in both amniotic fluid and blood provide new insights into fetal development and pathology. Prenatal diagnosis is poised to evolve from detection of aneuploidy to detection of deviation from normal development, which should provide novel opportunities for fetal treatment.

Gene expression analysis in pregnant women and their infants identifies unique fetal biomarkers that circulate in maternal blood

The discovery of fetal mRNA transcripts in the maternal circulation holds great promise for noninvasive prenatal diagnosis. To identify potential fetal biomarkers, we studied whole blood and plasma gene transcripts that were common to 9 term pregnant women and their newborns but absent or reduced in the mothers postpartum. RNA was isolated from peripheral or umbilical blood and hybridized to gene expression arrays. Gene expression, paired Students t test, and pathway analyses were performed. In whole blood, 157 gene transcripts met statistical significance. These fetal biomarkers included 27 developmental genes, 5 sensory perception genes, and 22 genes involved in neonatal physiology. Transcripts were predominantly expressed or restricted to the fetus, the embryo, or the neonate. Real-time RT-PCR amplification confirmed the presence of specific gene transcripts; SNP analysis demonstrated the presence of 3 fetal transcripts in maternal antepartum blood. Comparison of whole blood and plasma samples from the same pregnant woman suggested that placental genes are more easily detected in plasma. We conclude that fetal and placental mRNA circulates in the blood of pregnant women. Transcriptional analysis of maternal whole blood identifies a unique set of biologically diverse fetal genes and has a multitude of clinical applications.

Neonatal Salivary Analysis Reveals Global Developmental Gene Expression Changes in the Premature Infant

BACKGROUND There is an important need to develop noninvasive biomarkers to detect disease in premature neonates. Our objective was to determine if salivary genomic analysis provides novel information about neonatal expression of developmental genes. METHODS Saliva (50-200 microL) was prospectively collected from 5 premature infants at 5 time points: before, starting, and advancing enteral nutrition; at the introduction of oral feeds; and at advanced oral feeds. Salivary RNA was extracted, amplified, and hybridized onto whole-genomic microarrays. RESULTS Bioinformatics analyses identified 9286 gene transcripts with statistically significant gene expression changes across individuals over time. Of these genes, 3522 (37.9%) were downregulated, and 5764 (62.1%) were upregulated. Gene expression changes were highly associated with developmental pathways. Significantly downregulated expression was seen in embryonic development, connective tissue development and function, hematologic system development and function, and survival of the organism (10(-14) < P < 10(-3)). Conversely, genes associated with behavior, nervous system development, tissue development, organ development, and digestive system development were significantly upregulated (10(-11) < P < 10(-2)). CONCLUSIONS Comparative genomic salivary analyses provide robust, comprehensive, real-time information regarding nearly all organs and tissues in the developing preterm infant. This innovative and noninvasive technique represents a new approach for monitoring health, disease, and development in this vulnerable patient population. By comparing these data in healthy infants with data from infants who develop medical complications, we expect to identify new biomarkers that will ultimately improve newborn care.

Optimal techniques for mRNA extraction from neonatal salivary supernatant.

Background: Gene expression profiling of the salivary supernatant is emerging as a new and important source of real-time, systemic, biological information. However, existing technologies prevent RNA extraction of small quantities found in neonatal salivary supernatant. Objective: The aim of this study was to develop techniques to enhance extraction of cell-free RNA from neonatal salivary supernatant. Methods: Two saliva samples (10–100 µl) were serially collected from newborns (36–41 weeks’ gestation) (n = 13) and stabilized. Total RNA was extracted from salivary supernatant with the use of two modified extraction techniques: Qiagen RNAprotect® Saliva Mini Kit (method 1) and the QIAamp Viral RNA Mini Kit (method 2). Quantitative RT-PCR amplification for GAPDH was performed on extracted salivary samples. Statistical analyses were performed on mean threshold cycle (Ct) levels to compare RNA yield from each protocol. Paired microarray analyses were made between neonatal whole saliva and supernatant (n = 3) to discern gene expression differences between these biolayers. Results: mRNA was successfully extracted and amplified from all salivary supernatant samples. Extraction with method 2 yielded more RNA than with method 1 (p = 0.008). There was a 7.5% discordance between paired gene expression analyses for whole saliva and supernatant. Genes that were statistically significantly upregulated in supernatant highlighted 16 distinct biological functions not seen in whole saliva. Conversely, only two biological functions were unique to whole saliva. Conclusion: Neonatal cell-free salivary supernatant mRNA may be readily extracted and utilized on downstream applications. These technical enhancements allow for further exploration of the diagnostic potential of the neonatal salivary transcriptome.

Neuropeptide Y2 receptor (NPY2R) expression in saliva predicts feeding immaturity in the premature neonate.

Background The 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 Findings Salivary 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/Significance Developmental 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.

Fetal nucleic acids in maternal body fluids: an update.

Abstract:  Our laboratory continues to be actively involved in the development of new biomarkers for prenatal diagnosis using maternal blood and amniotic fluid. We have also developed a mouse model that demonstrates that cell‐free fetal (cff) DNA is detectable in the pregnant maternal mouse. In human maternal plasma and serum we have analyzed factors that are important in the clinical interpretation of cff DNA levels. Maternal race, parity, and type of conception (natural or assisted) do not affect cff DNA levels, but maternal weight does. We have also analyzed the relationship between placental volume, using a three‐dimensionsal ultrasound examination, and cff DNA levels. Surprisingly, there is no association between these values. Finally, we are using specific disease models (such as congenital diaphragmatic hernia and twin‐to‐twin transfusion) to understand the effects of gestational age and specific pathology on fetal gene expression by analyzing cell‐free mRNA levels in maternal plasma. In the amniotic fluid we have focused on improvements in recovery of cff DNA and mRNA. By optimizing recovery we have made some interesting observations about differences in fetal DNA between blood and amniotic fluid. In addition, we have successfully hybridized cff DNA in amniotic fluid to DNA microarrays, permitting assessment of fetal molecular karyotype. We also have preliminary data on fetal gene expression in amniotic fluid. Finally, we remain actively involved in promoting noninvasive prenatal testing in the United States, such as encouraging the use of fetal DNA for fetal rhesus D assessment. On the other hand, we are cautious and concerned about the accuracy of “at‐home” kits for fetal gender detection.

Detection and Potential Utility of C-Reactive Protein in Saliva of Neonates

Objective: We aimed to detect C-reactive protein (CRP) in neonatal saliva and evaluate its diagnostic utility. Study Design: Salivary and serum samples (n = 89) were collected from 40 neonates. Salivary CRP levels were determined using an enzyme-linked immunosorbent assay; serum CRP was measured per hospital protocol. Correlation coefficients with 95% confidence intervals and robust linear regression measured association while receiver–operator characteristic curves described the accuracy of salivary CRP in discriminating abnormal serum CRP thresholds of ≥10 and 5 mg/L. Corresponding sensitivities and specificities were calculated for these salivary cutpoints. Results: The area under the curve for salivary CRP in predicting serum CRP levels of ≥10 and 5 mg/L were 0.81 and 0.76, respectively. The corresponding sensitivity and specificity for raw salivary CRP to discriminate a serum CRP of ≥5 mg/L was 0.54 and 0.95, respectively. The corresponding sensitivity and specificity for raw salivary CRP to discriminate a serum CRP of ≥10 mg/L was 0.64 and 0.94, respectively. A statistically significant correlation was observed between serum and salivary CRP (r = 0.62, p < 0.001). Conclusion: C-reactive protein is detectable in neonatal saliva and can predict abnormal serum CRP thresholds. Salivary CRP analysis represents a feasible screening tool for detecting abnormal serum CRP levels.

Computational Gene Expression Modeling Identifies Salivary Biomarker Analysis that Predict Oral Feeding Readiness in the Newborn

OBJECTIVE To combine mathematical modeling of salivary gene expression microarray data and systems biology annotation with reverse-transcription quantitative polymerase chain reaction amplification to identify (phase I) and validate (phase II) salivary biomarker analysis for the prediction of oral feeding readiness in preterm infants. STUDY DESIGN Comparative whole-transcriptome microarray analysis from 12 preterm newborns pre- and postoral feeding success was used for computational modeling and systems biology analysis to identify potential salivary transcripts associated with oral feeding success (phase I). Selected gene expression biomarkers (15 from computational modeling; 6 evidence-based; and 3 reference) were evaluated by reverse-transcription quantitative polymerase chain reaction amplification on 400 salivary samples from successful (n = 200) and unsuccessful (n = 200) oral feeders (phase II). Genes, alone and in combination, were evaluated by a multivariate analysis controlling for sex and postconceptional age (PCA) to determine the probability that newborns achieved successful oral feeding. RESULTS Advancing PCA (P < .001) and female sex (P = .05) positively predicted an infants ability to feed orally. A combination of 5 genes, neuropeptide Y2 receptor (hunger signaling), adneosine-monophosphate-activated protein kinase (energy homeostasis), plexin A1 (olfactory neurogenesis), nephronophthisis 4 (visual behavior), and wingless-type MMTV integration site family, member 3 (facial development), in addition to PCA and sex, demonstrated good accuracy for determining feeding success (area under the receiver operator characteristic curve = 0.78). CONCLUSIONS We have identified objective and biologically relevant salivary biomarkers that noninvasively assess a newborns developing brain, sensory, and facial development as they relate to oral feeding success. Understanding the mechanisms that underlie the development of oral feeding readiness through translational and computational methods may improve clinical decision making while decreasing morbidities and health care costs.

Great Expectorations: The Potential of Salivary “Omic” Approaches in Neonatal Intensive Care

Among those that require critical care, preterm neonates have the greatest limitations on available blood or body fluids for clinical or research-based assessments. Recent technological advancements have improved our ability to detect genetic, proteomic and microbial material at the nanoscale level, making analyte and biomarker assessment from even the smallest quantities possible. Saliva is a unique body fluid that not only may be noninvasively and repeatedly obtained, but also contains multiple serum components, making it promising for noninvasive assessment of the newborn. The integration of high-throughput or ‘omic’ approaches on neonatal saliva holds great potential to improve diagnostic and prognostic accuracy for a wide range of developmental and pathological conditions affecting the vulnerable preterm neonatal population. Herein, we review the clinical applications and technical considerations regarding the integration of salivary ‘omic’ technology into the neonatal intensive care unit.

Insights Into Fetal and Neonatal Development Through Analysis of Cell-Free RNA in Body Fluids

The use of cell-free nucleic acids in the circulation of pregnant women for noninvasive prenatal diagnosis is arguably one of the hottest current topics in prenatal medicine. Between 1997 and the present era this field has gone from basic research to clinical application for diagnosis of fetal gender and Rhesus D status. Over the next few years it is likely that noninvasive prenatal diagnosis for Down syndrome will also be possible. Here we summarize current and future clinical applications of analyzing cell-free fetal DNA and RNA in both maternal and neonatal body fluids, including maternal plasma, serum, whole blood, amniotic fluid, and neonatal saliva. We describe methods to evaluate normal and abnormal fetal and neonatal development using gene expression microarrays. We also discuss the ways in which differentially-regulated gene lists can advance knowledge of both fetal and neonatal biology, as well as suggest novel possibilities for fetal and neonatal treatment.