Diagnostic / Prognostic CIFASD Strategic Objective: ‘Clinically-useful diagnostic criteria for FASD’ FACIAL MORPHOLOGY Dysmorphology Core 3D Camera Study BRAIN STRUCTURE sMRI / DTI NEUROBEHAVIOR “BIOMARKERS” Diagnostic / Prognostic BRAIN FUNCTION fMRI / MEG 3

Biomarkers of Ethanol Exposure / Effect? MCV, GGT, CDT ( blood ) FAEEs ( meconium, hair? ) Ethyl Glucuronide ( blood, urine, hair? ) “Novel Biomarkers” ( blood?, placenta? ) Genomics Proteomics Metabolomics microRNAs 3

Prenatal Ethanol Exposure Paradigm VOLUNTARY DRINKING - 5% Ethanol in 0.066% saccharin water - Two-bottle choice paradigm - Intermittent consumption (4 hr/day) - Peak maternal serum EtOH: Long-Evans rats: 84 mg / dL Black 6/J mice: 125 mg / dL - Saccharin water, volume-matched control 3

Altered Gene Expression Cannabinoid Receptor 1 S100 Calcium Binding Protein A4 Galanin Receptor 2 Integrin Alpha 7 Gremlin 1 S100 Calcium Binding Protein G Hemoglobin Epsilon 1 Secreted Frizzle-related Protein 4 Matrix Metalloproteinase 2 S100 Calcium Binding Protein B Deiodinase Iodothyronine 2 Lipopolysaccharide Binding Protein Galanin Hemoglobin Delta Toll-like Receptor 4 Placental Growth Factor Transforming Growth Factor Alpha Heart & Neural Crest Derivative 2 11b-Hydroxysteroid Dehydrogenase 2 Transient Receptor Potential Cation Channel 4 Serine Peptidase Inhibitor, Kazal Type 5 EGF-containing Fibulin-like Extracellular Matrix Protein 1 3

2D PAGE Analysis of Placental Proteins – 150 mg samples Control Ethanol  pH  mw  Courtesy of Prof. Thierry Jouenne & colleagues, University of Rouen

Role of Animal Models in Biomarker Development Rodents - Rapid, cost-effective identification of candidates - Ethanol-dose, timing, frequency characterizations - Other “risk factor” interaction assessments (nicotine, stress, malnutrition, heavy metals, etc.) - Correlation with behavioral / physiologic outcomes Sheep Model - Validation of biosignature patterns - Functional Implications 3

Biomarker Development Strategy Preclinical Studies Clinical Studies Identification Maternal Data & Tissue Banking Characterization Offspring Measures Validation Biomarker Assessment 3

Consensus Protocol Information / Sample Collection Interview: TWEAK, AUDIT, Maternal Information Maternal: Blood, Urine, Hair Ultrasound (including Doppler) Placenta, Cord Blood Neonate: Blood, Meconium, Hair? Neurobehavioral Data 3

Consensus Protocol Information / Sample Collection Times First Prenatal Visit: (1st trimester) Follow-up Visit: (3rd trimester) Delivery: Follow-up: (6 to 12 months of age) 3

Prospective Human Biomarker Study Sites PASS France CIFASD - Ukraine SDSU/UCSD, UCLA, Emory, UNM, others ? Other International Sites ? South Africa Finland Italy Canada Sweden National Children’s Study New Mexico, California, Maryland, New York, others? 3

Decreased Protein Expression Altered Gene Expression Cannabinoid Receptor 1 S100 Calcium Binding Protein A4 Galanin Receptor 2 Integrin Alpha 7 Gremlin 1 S100 Calcium Binding Protein G Hemoglobin Epsilon 1 Secreted Frizzle-related Protein 4 Matrix Metalloproteinase 2 S100 Calcium Binding Protein B Deiodinase Iodothyronine 2 Lipopolysaccharide Binding Protein Heart & Neural Crest Derivative 2 Transient Receptor Potential Cation Channel 4 Galanin Serine Peptidase Inhibitor, Kazal Type 5 Hemoglobin Delta Toll-like Receptor 4 Transforming Growth Factor Alpha Placental Growth Factor 11b-Hydroxysteroid Dehydrogenase 2 EGF-containing Fibulin-like Extracellular Matrix Protein 1 Decreased Protein Expression Proteasome Subunit Beta Type 3 Adenine Phosphoribosyl Transferase Keratin 18 Desmin Enolase Alpha 1 Valosin-containing Protein Protein Disulfide Isomerase Eukaryotic Translation Elongation Factor 2 Stomatin Peroxiredoxin 5 Precursor Chaperon Protein TCP1 Subunit 2 3-Hydroxyisobutyrate Dehydrogenase 3

Long-Term Strategy Preclinical Clinical Biomarkers Biomarkers Fetal / Neonatal Brain Effects Weanling Behaviors & Neurophysiology Neonatal Behaviors & Neurophysiology 3