Ukraine Clinical Project Progress April 7-8, 2014

Aims 1 & 2 Recall mothers and children for visits at 2 yrs and 3.5-5 yrs of age Interviews Growth Food records Biological sampling 3D imaging Cardiac orienting response paradigm Preschool testing battery at 3.5-5 yr visit

Aims 1 & 2 Recruit an additional 120 pregnant women with same follow-up as Phase II Incorporate these children/mothers into the Phase III protocol

Aims 1 & 2: Phase III Visits   Visit Under Phase III Protocol Number of Visits Number of Unique Subjects New pregnant women enrolled Phase III 137 Mothers re-consented in Phase III for new data collection 42 Dysmorphology exams completed 325 223 Prenatal ultrasounds completed 240 139 Cardiac orienting paradigm completed 205 113 3D images captured 72 2 year old visits completed 17 3.5-5 year old visits completed 25

On-Site Training Ukrainian Staff on Administration and Scoring of Preschool Test Protocol. Developed Preschool Battery for Assessment of Cognitive and Behavioral Effects of Prenatal Alcohol Exposure Sensitive to Effects of Prenatal Exposure and Consistent with CIFASD Neurobehavioral Battery Suitable for Children 3 ½ to 5 years Not reliant on Verbal Skills to avoid Language Confounds Acceptable to Ukrainian Staff and Research Participants Perceived by Ukrainian colleagues to have clinical merit Assuring Reliability and Validity in Test Administration and Scoring. Repeated On Site Training Review of records of Test Administrations Regular Skype “conferences”

Brain Involvement in Orienting Responses The heart receives input from the 10th cranial nerve via the medulla oblongata Electrical stimulation in animal (rabbit) models of the vmPFC (ventral-medial prefrontral cortex) and DLPFC (dorsal lateral prefrontal cortex) result in ORs Mediodorsal nuclei of the thalamus and the intralaminar nucleus of the thalamus are also involved in OR responses (Powel, Watson, & Buchanan, 1990) Cardiac orienting responses index early frontal lobe functioning

Improvements in the New Neurophysiological Protocol Expanded to 2 sites Upgraded equipment to Biopac’s Wireless Bioharness and trained staff to carry out the protocol

Preschool Physiology Protocol Repeating Auditory OR Response (5/3) Three Little Pigs (Auditory/Visual Habituation Combined 3/3) Auditory stimuli: Habituation: 400-1000 hertz pure tone pairs presented contiguously for 12 sec each Dishabituation: 700-1000 hertz pure tone pairs presented contiguously for 12 sec each

Reaction Time Task Looks at sustained attention while performing a reaction time task. During sustained attention HR and HR variability is reduced Children press a special preschool mouse when they see a picture

Discriminate Function Analysis-Combining All ORs Auditory Future Goals Determine predictive validity to 12-months Bayleys Determine predictive validity to preschool period Improve mathematical modeling of the dynamic OR response and determine if this improves prediction Discriminate Function Analysis-Combining All ORs 6-Month Classification Predicted Normal Delay 91 9 2 10 90.2 % Classified Correctly Specificity .91 (% of persons without delay who test negative) Sensitivity .83 (% of persons with delay who test positive) Habituation Dishabituation

Aim 3: miRNA 142 archived maternal plasma samples selected and shipped to Texas A&M 71 mothers in 3 groups sampled: two available maternal blood samples per subject at least one physical exam of the child at least one completed neurobehavioral test samples are classified into three groups exposed mothers who had children with at least one physical feature of FASD and at least one Bayley score <85 exposed mothers who had children with no physical features of FASD and no Bayley score <85 unexposed/low exposed mothers

Aim 3: miRNA Initial quality control assessment of samples has been performed for 60 plasma samples and deemed adequate. Dr. Miranda’s lab has isolated RNA from a total of 24 samples and has been able on average to recover ~1000 ng RNA per sample There are no statistically significant differences in RNA recovery due to recruitment site, stage of pregnancy or treatment group

A B p<0.04 Plasma Erythrocytes Human Plasma Samples Plasma Peaks TCR1944 3rd Trimester TCR1944 1st Trimester Sheep Free Hemoglobin Deoxy- and oxy-hemoglobin Plasma Erythrocytes

Aim 4: Genetics/Epigenetics 500 WBC maternal samples banked at UCSD for assessment of methylation status Selecting samples based on criteria for miRNA and likelihood that paired child sample will be available Plan to process all selected samples in one batch

Aim 4: 3D images (one site) Exposed Low/Unexposed 6 months 20 12 months 3 7 2 years 2 11 4 years 4 5 Total 29 43

Aim 4: Telemedicine See Dysmorphology Core

Phase II Analyses Dr. Keen UC Davis has finished analysis of all 1,077 samples from the 10 shipments for Zn, Cu, Fe, Mg, Ca, transferrin receptor and ceruloplasmin activity Samples through shipment 9 have been analyzed for homocysteine, C-reactive protein, ferritin and vitamin D Choline and metabolites analyses have been completed for 802/1077 samples (74.5%)

Phase II Analyses: Dose (Number Binge Days) at Conception vs Phase II Analyses: Dose (Number Binge Days) at Conception vs. Features FASD   Feature Control N = 256 0 days/wk n =71 1 day/wk n = 82 2 days/wk n = 39 3+ days/wk n = 24 Four-group Alcohol Exposed p-value Five-group PF <10th 20 (7.8%) 15 (21.1%) 17 (21.0%) 12 (30.8%) 6 (25.0%) 0.640 <0.001 Smooth philtrum 28 (10.9%) 10 (14.1%) 8 (9.8%) 6 (15.4%) 7 (29.2%) 0.139 0.104 Thin vermilion 48 (18.8%) 18 (25.4%) 10 (12.2%) 11 (45.8%) 0.003 0.002 OFC <10th 14 (5.5%) 7 (9.9%) 12 (14.6%) 8 (20.5%) 0.116 FAS Dx on features 5 (2.0%) 7 (10.0%) 3 (3.7%) 5 (12.8%) 5 (20.8%) 0.055 6M MDI <85 28/205 (13.7%) 16/56 (28.6%) 18/64 (24.1%) 10/25 (40.0%) 13/24 (54.2%) 0.097 6M PDI <85 47/204 (23.0%) 18/56 (32.1%) 24/64 (37.5%) 9/24 0.893 0.079

Phase II Analyses: Dose (Drinks per Week) Mid-Pregnancy vs Phase II Analyses: Dose (Drinks per Week) Mid-Pregnancy vs. Features FASD Feature Control N = 256 <3 drinks/wk n = 182 3-7 drinks/wk n = 16 8+ drinks/wk Three-group Alcohol Exposed p-value   Four-group PF <10th 20 (7.8%) 32 (17.7%) 6 (37.5%) 12 (75.0%) <0.001 Smooth philtrum 28 (10.9%) 21 (11.5%) 3 (18.8%) 7 (43.8%) 0.004 Thin vermilion 48 (18.8%) 36 (19.8%) 5 (31.2%) 10 (62.5%) OFC <10th 14 (5.5%) 24 (13.2%) FAS Dx on features 5 (2.0%) 8 (4.4%) 9 (56.2%) 6M MDI <85 28/205 (13.7%) 38/142 (26.8%) 7/12 (58.3%) 10/13 (76.9%) 6M PDI <85 47/204 (23.0%) 46/142 (32.4%) 6/12 (50.0%) 9/13 (69.2%) 0.020

Alcohol Low/Unexposed Group Phase II Analyses: Brain Ultrasound Measures as Predictors of 6 month Bayley Scores Bayley 6 Mos Ultrasound Measure N Alcohol Exposed Group Alcohol Low/Unexposed Group Estimate SE p-value MDI TCD 105 -0.07 0.65 0.916 172 0.11 0.34 0.746   OFD 104 0.43 0.23 0.069 171 -0.05 0.14 0.723 CCD 103 -0.47 0.45 0.300 0.40 0.080 FTD 0.51 0.21 0.018 0.47 0.13 <0.001 OOD 0.48 0.326 170 0.67 0.26 0.010 IOD -1.22 0.46 174 -1.12 OD 1.21 0.85 0.159 -0.26 0.52 0.614 Each measure adjusted for gestational age at ultrasound, and each model for one or more of the following that met criteria as a confounder: maternal age, parity, socioeconomic status, education, pre—pregnancy BMI, vitamin use and smoking

Publication Progress Chambers et al, Alcoholism Clinical Experimental Research, accepted 9/2013

for vitamin use in 1 mo before Alcohol use 3 mo prior to pregnancy and multivitamin use 1 mo prior in 111,644 U.S. women in PRAMS 2004-8 Alcohol in 3 mo before pregnancy OR for vitamin use in 1 mo before 95% CI p for trend Number of drinks in average week None <=3 4-6 7-13 >=14 1.00 0.76 0.60 0.49 0.39 0.72, 0.80 0.56, 0.66 0.44, 0.55 0.32, 0.47 <0.001 Number of binge episodes (5 or more) 1 2-3 4-5 >=6 0.66 0.56 0.50 0.71, 0.81 0.62, 0.70 0.50, 0.64 0.44, 0.56 Weiss LA & Chambers CD, Alcohol Clin Exp Res, 2013;37:1595-600; adjusted for education, race/ethnicity, income, age, parity

Publications Progress Balaraman S, Lunde ER, Sawant O, Cudd TA, Washburn SE and Miranda RC. Maternal and Neonatal Plasma MicroRNA Biomarkers for Fetal Alcohol Exposure in an Ovine Model. Alcoholism: Clinical and Experimental Research, 2014 epub Uriu-Adams JY, Obican SG, and Keen CL. Vitamin D and maternal and child health: Overview and implications for dietary requirements. Birth Defects Research. Part C: Embryo Today, 2013;99:24-44

Plans Accelerate recruitment of children for the 2 and 3.5-5 year old visits in the Spring and Summer months in Rivne and Khmelnytsky to meet recruitment objective of 90 children per group in July, 2014 Perform initial analysis on quality of biological samples from children Generate preliminary results from Aim 3 and the genetic/epigenetic portion of Aim 4 Provide additional 3D images to Drs. Foroud and Hammond by moving the camera from site to site

Plans Continue in progress submissions for publication Baseline minerals Baseline vitamins Baseline methyl donors Intervention: 6 month and 12 month analyses Pre/postnatal growth trajectory by maternal nutritional status Ultrasound findings Physical features in relation to dose Retinol levels at baseline and inner canthal distance Continue planned additional publications