SBIR-STTR Award

Prenatal alcohol exposure is the leading preventable cause of birth defects in the United States, producing an array of neurological, behavioral and physical abnormalities collectively known as fetal alcohol spectrum disorders (FASD). Early intervention and treatment programs have been shown to reduce some of the long-term adverse effects, but this is dependent on identifying children at risk of fetal damage which is not detected in most cases. Many children affected by FASD do not exhibit obvious physical indications of prenatal alcohol-associated birth defects, creating challenges in making timely and accurate diagnoses. There is a critical need for development of biomarkers that can detect babies heavily exposed to alcohol throughout pregnancy, particularly during the first trimester when the fetal brain is most vulnerable to teratogens, to predict those at risk for developing the neurobehavioral and developmental disabilities associated with FASD. Prenatal alcohol exposure has been shown to alter DNA methylation patterns of genes known to play important roles in development and contribute to abnormal embryonic development, suggesting that alcohol-associated alterations in DNA methylation profiles, identified from neonatal blood spot samples, could be used as an alcohol biomarker in neonates. Coupling the technology United States Drug Testing Laboratories has developed for detection of the direct alcohol biomarker phosphatidylethanol (PEth) in blood spots with screening epigenetic changes associated with prenatal alcohol exposure provides an innovative approach to examine novel biological markers of alcohol exposure occurring throughout pregnancy that may be predictive of fetal damage. The objective of this Phase I project is to determine the feasibility of using neonatal blood spots to screen alcohol-induced DNA methylation alterations and establish whether these epigenetic modifications correlate with detection of PEth. The selection of candidate regions of differential methylation will be determined by whole-genome methylation scanning of DNA from neonatal blood spot samples collected from infants with known exposure to alcohol in utero and comparing them with non- exposed controls. If prenatal alcohol exposure is significantly associated with a defined set of differentially methylated DNA sites, the feasibility of using epigenetic markers to detect newborns at risk of fetal damage will have been established and these epigenetic patterns could be further examined as possible predictive biomarkers of FASD. Development and implementation of a quantitative DNA methylation screening assay, to analyze sites of differential methylation identified in this Phase I study, would be the goal of a Phase II study.

Public Health Relevance Statement:


Public Health Relevance:
More than 80,000 babies born in the United States each year are at high risk for alcohol- induced birth defects, making prenatal alcohol exposure the foremost preventable source of birth defects. Early intervention and treatment programs have been shown to reduce some of the long-term adverse effects, but this is dependent on identifying children at risk of fetal alcohol damage which is not possible in most cases. This project will establish the feasibility of using epigenetic markers, identified from neonatal blood spot samples, to detect newborns at risk of fetal damage associated with excessive prenatal alcohol exposure.

NIH Spending Category:
Alcoholism; Brain Disorders; Conditions Affecting the Embryonic and Fetal Periods; Fetal Alcohol Syndrome; Genetic Testing; Genetics; Human Genome; Intellectual and Developmental Disabilities (IDD); Pediatric; Perinatal Period - Conditions Originating in Perinatal Period; Prevention; Substance Abuse

Project Terms:
Adult; Adverse effects; Affect; Alcohol abuse; alcohol exposure; alcohol screening; Alcohols; Analytical Toxicology; base; Behavioral; Biological Assay; Biological Markers; Birth; Blood; Brain; candidate selection; Child; Congenital Abnormality; Coupling; Detection; Development; Developmental Disabilities; Diagnosis; DNA; DNA analysis; DNA Methylation; drug testing; Early Intervention; Early treatment; effective intervention; effective therapy; Embryonic Development; epigenetic marker; Epigenetic Process; Exhibits; fetal; Fetal Alcohol Exposure; Fetal Alcohol Spectrum Disorder; fetal drug exposure; First Pregnancy Trimester; Genes; Genetic Screening; Genome; Goals; high risk; in utero; Incidence; Infant; innovation; Laboratories; medical specialties; Methods; Methylation; minimally invasive; Modification; Neonatal; Neonatal Alcohol Exposure; Neonatal Screening; neonate; neurobehavioral; Neurologic; Newborn Infant; novel; Outcome; Pattern; Phase; phase 1 study; phase 2 study; phosphatidylethanol; Play; Pregnancy; prenatal; Prevalence; programs; public health relevance; Research; research and development; Risk; Role; Sampling; Scanning; Schools; screening; Site; Source; Spottings; Substance Abuse Detection; Technology; Teratogens; Time; treatment program; United States

Prenatal alcohol exposure is the leading preventable cause of birth defects in the United States, producing an array of neurological, behavioral and physical abnormalities collectively known as fetal alcohol spectrum disorders (FASD). Early intervention and treatment programs have been shown to reduce some of the long-term adverse effects, but this is dependent on identifying children at risk of fetal damage which is not detected in most cases. Many children affected by FASD do not exhibit obvious physical indications of prenatal alcohol-associated birth defects, creating challenges in making timely and accurate diagnoses. There is a critical need for development of biomarkers that can detect babies heavily exposed to alcohol throughout pregnancy to predict those at risk for developing the neurobehavioral and developmental disabilities associated with FASD. Prenatal alcohol exposure has been shown to alter DNA methylation patterns of genes known to play important roles in development and contribute to abnormal embryonic development, suggesting that alcohol-associated alterations in DNA methylation profiles, identified from neonatal blood spot samples, could be used as an alcohol biomarker in neonates. Findings from our Phase I study demonstrate that epigenetic profiles, specifically the differences in site-specific CpG DNA methylation, can differentiate the incidence of prenatal alcohol exposure using neonatal blood spots. The proposed SBIR Phase II project seeks to develop and validate the technology to generate quantitative DNA methylation screening assays using bisulfite pyrosequencing to analyze the differentially methylated sites that were found to be significantly associated with prenatal alcohol exposure. The technology that we will develop will use samples that are already routinely collected from newborns for comprehensive genetic screening assays (heel stick blood spots) and more recently for prenatal alcohol exposure screening (umbilical cord and heel stick blood spots) to 1) extract and purify genomic DNA using an automated system; 2) perform bisulfite-PCR reactions; and 3) screen for epigenetic DNA methylation biomarkers of prenatal alcohol exposure using pyrosequencing. These assays will be used to screen genomic DNA collected from a large sampling of PEth positive and PEth negative neonatal blood spots to identify the epigenetic modifications that most significantly correlate with fetal alcohol exposure. Our long-term goal is to examine whether there is a correlation between these epigenetic biomarkers and an increased risk for the developmental disabilities associated with FASD.

Public Health Relevance Statement:
PROJECT NARRATIVE The prevalence of FASD is now estimated to be as high as 2 to 5% of school children, making prenatal alcohol exposure the most preventable source of birth defects in the United States. Early intervention and treatment programs have been shown to reduce some of the long-term adverse effects, but this is dependent on identifying children at risk of fetal alcohol damage which is not possible in most cases. The proposed Phase II project will develop and validate the technology to run quantitative DNA methylation screening assays using bisulfite pyrosequencing from newborn dried blood spot samples to analyze differentially methylated sites that were shown to be significantly associated with prenatal alcohol exposure in the Phase I study with the goal of validating an epigenetic biomarker of prenatal alcohol exposure.

Project Terms:
accurate diagnosis; Adverse effects; Affect; alcohol exposure; Alcohols; Behavioral; Biological; Biological Assay; Biological Markers; biomarker development; Birth; bisulfite; Blood; Blood specimen; Child; cohort; Congenital Abnormality; Detection; Development; Developmental Disabilities; Diagnosis; DNA; DNA Methylation; drug testing; Early Intervention; effective intervention; effective therapy; Embryonic Development; epigenetic marker; Epigenetic Process; fetal; Fetal Alcohol Exposure; Fetal Alcohol Spectrum Disorder; fetal drug exposure; Genes; Genetic Screening; Genomic DNA; Genomics; Goals; Heel; High birth weight infant; High Prevalence; in utero; Incidence; intervention program; Laboratories; Magnesium Chloride; medical specialties; Methods; Methylation; methylation biomarker; methylation pattern; Modification; Neonatal; Neonatal Screening; neonate; neurobehavioral; Neurologic; Newborn Infant; Outcome; Pediatric Hospitals; Phase; phase 1 study; phosphatidylethanol; Play; Pregnancy; prenatal; prenatal exposure; Prevalence; Program Development; prospective; pyrosequencing; Reaction; Research; research and development; Risk; Role; Running; Sampling; Schools; screening; Site; Small Business Innovation Research Grant; Source; Spottings; System; Technology; Temperature; Testing; Time; Toxicology; treatment program; Umbilical Cord Blood; Umbilical cord structure; United States; Uruguay; Validation; West Virginia; whole genome; Woman