Ukraine Clinical Study: U01 Christina Chambers, PhD Carl Keen, PhD Claire Coles, PhD Julie Kable, PhD Kenneth Lyons Jones, MD Wladimir Wertelecki, MD Rajesh Miranda, PhD Kelly Frazer, PhD Rhongui Xu, PhD Lyubov Yevtushok, MD Natalya Zymak, MD

Specific Aim 1 Determine if the beneficial effects of a prenatal multivitamin/mineral supplement in alcohol-exposed children persists to pre-school age Does the effect of the nutrient supplements persist in children who are 3.5-4.5 years of age? Do measures previously collected in infancy correlate with preschool measures?

Specific Aim 2 Determine if prenatal alcohol exposure affects child nutritional status, and determine to what extent that affects child growth and performance Does prenatal exposure affect current child nutritional status in context of child’s dietary intake as measured between 2 and 5 years of age? Does child’s current nutritional status predict neurodevelopmental performance and/or growth through 5 years of age in the context of prenatal alcohol exposure?

Specific Aim 3 Determine if a miRNA biomarker for alcohol exposure is useful as a marker of recent or distant exposure to alcohol in the human Can an alcohol-related maternal circulating miRNA profile be identified among drinking women? Does that profile (or another) persist in 3rd trimester maternal plasma, and/or in the child? Do any miRNA profiles identified in mother or child relate to FASD (growth and performance)?

Specific Aim 4 Explore genetic and epigenetic risk factors for FASD, effect of maternal/child nutrition on 3D facial images, and telemedicine approaches to earlier diagnosis Do genetic variants suggested by other CIFASD collaborators occur more frequently in heavily exposed mother/child pairs? Is variance in methylation status associated with heavily exposed mother/child pairs in context of nutritional status? Is 3D imaging an effective diagnostic aid in infants/young children?Does pre or postnatal nutrition impact image classification? Can telemedicine approaches in remote areas aid in earlier/accurate diagnosis of FASD?

Sample Retain and test ~300 children from original sample with preschool battery Continue to perform Bayleys and heart rate monitoring for children up to12 months of age Recruit and follow 120 new pregnant women with no MVM intervention Continue blood sampling 2x in pregnancy for nutritional status, and prenatal ultrasounds for newly recruited women

Progress 2 U.S. (Emory and Davis) and 1 foreign subcontract all in place 1 U.S. subcontract for Year 2 (Texas A&M) in progress Collaborator’s agreement terms negotiated for administrators at both foreign sites Purchase of and translation into Ukrainian of preschool neurobehavioral testing battery Training in person for both psychologists in Ukraine initiated October, 2011 Purchase of new (portable) heart rate monitoring setup in progress; replacement of existing non-functional equipment at the same time; both to be delivered March, 2013

Progress Obtained IRB approval to use archived maternal blood samples for de-identified analysis (Pediatric Genomics at UCSD; Rutgers; Texas A&M) Selecting appropriate samples Oragene kits for saliva collection delivered to Ukraine Protocol for child blood and urine collection reviewed with Ukrainian partners and modified to add a third visit to reduce per visit blood quantity required and to improve retention Adding a local test to provide immediate feedback to participants Customs process for 3D camera to be initiated Jan, 2013 Created 15 minute training video for new sample preparation

Progress Informatics group working with U.S. Data Manager and Julie Kable to finalize the neurobehavioral input tool Created an Access database for complex scheduling of visits and a user manual; trained Khmelnytsky Data Manager on this in December

Enrollment Data Shows the entire history of the subject, e.g. group, due date, exam date, any LTF

Criteria for eligible subject current age greater than 3 years old but less than 6yr &11mo not LTF/drop Status on 12 month (and previous visits)

Preschool evaluation eligible subject list Setting the criteria from the previous slide will give us this list

Preschool cumulative list An overview of all subjects who have reached testing age and the test status (Status)

Cumulative counts by ETOH/Vit group Can look at the cumulative number of subjects eligible and/or tested by ETOH group and by Vit group ETOH Group (0,1,2) Row sum Counts of subjects by Status, site, ETOH Group, and Vit Group

Progress: data analysis DSMB met in 2012 and approved formal interim data analysis Revised statistical analysis plan (approved by the DSMB) called for nominal p-value cutoff of <0.005 with sample tested at 12 months (50 each in the no MVM intervention and yes MVM intervention alcohol-exposed group); and revised nominal p-value cutoff for final analysis of 0.048 in order to ensure an overall 2-sided Type I error rate of 0.05 Due to 3 sets of twin pairs in the MVM intervention group, sample had to be reduced by randomly selecting one of each set

Progress: interim analysis BSID scores at 12m by nutrition intervention in alcohol exposed Group Vitamin No N=50 Yes P-value PDI 95.1 (16.5) 98.5 (12.7) 0.255 MDI 88.0 (14.4) 93.7 (11.6) 0.032

Progress: Interim analysis: balanced by randomization groups Vitamin No Vitamin Yes Mean Age Years 25 Mean GA Weeks 20 17 Mean Parity .96 .91 Mean Years Education 13 Intervention Randomization None 46% MVM only 54% MVM+choline Smoker (%) Never Previous Quit in Pregnancy Continued in Pregnancy 27 8 42 23 34 10 30 26 Other Vitamin Use Some 73 32 68

Progress: final analysis Expect to have required 150 subjects tested at 12 months and data entered by Feb. 1 Will allow for all subsequent analyses pending publication of the primary analysis to proceed.

Progress: PRAMS publication: analysis of multivitamin supplement use and alcohol consumption 2004-2008 Phase 5 core questionnaire 111,644 women from 39 states who completed questions on Alcohol consumption in the 3 months before pregnancy Multivitamin or prenatal vitamin supplement use in the month before pregnancy

Progress: PRAMS analysis of multivitamin supplement use and alcohol consumption Drinks 3 Mo Before Pregnancy Multivitamin Use Adjusted OR 95% CI None 1.00 reference Up to 3 per week 0.76 0.72, 0.80 4-6 per week 0.60 0.56, 0.66 7-13 per week 0.49 0.44, 0.55 14 or more per week 0.39 0.32, 0.47 P-for-trend <0.001 Adjusted for maternal education, race/ethnicity, household income, age, parity

Progress: PRAMS analysis of multivitamin supplement use and alcohol consumption Number Binges 5 or more in 3 Mo Before Pregnancy Multivitamin Use Adjusted OR 95% CI None 1.00 reference 1 time 0.76 0.71, 0.81 2-3 times 0.66 0.62, 0.70 4-5 times 0.56 0.50. 064 6+ times 0.50 0.44, 0.56 P-for-trend <0.001 Adjusted for maternal education, race/ethnicity, household income, age, parity

Progress: PRAMS analysis of multivitamin supplement use and alcohol consumption

Progress: PRAMS analysis of multivitamin supplement use and alcohol consumption

Prenatal ultrasound brain measures - Ukraine sample Transverse Cerebellar Diameter Occipital Frontal Diameter Caval-Calvarial Distance Frontothalamic Distance Outer Orbital Distance Interorbital Distance Orbital Distance TCD T CCD OOD FTD

Progress: ultrasound papers (1): ultrasound measures at 4-6 months gestation as predictors of exposure Exposed N = 104 Unexposed N = 135 P-value* Frontothalamic distance 37.7 (5.4) 39.1 (4.7) 0.026 Caval calvarial distance 24.0 (3.1) 24.8 (3.0) 0.006 *Adjusted for gestational age, smoking, and vitamin use

Progress: ultrasound papers (1): ultrasound measures at 4-6 months gestation as predictors of physical features Feature U/S Measure Direction of U/S Measure Short pf’s Inter-orbital distance wider Thin vermillion narrower Smooth philtrum Occipital frontal diameter longer Maxillary arc Orbital diameter shorter Mandibular arc

Progress: ultrasound papers (2): ultrasound measures at 4-6 months gestation as predictors of Bayley score Examined MDI and PDI at 6 and 12 months in relation to the following measures (standardized for gestational age at measure), adjusted for alcohol group, vitamin group, alc x vit interaction, use of non-study vitamins, smoking status, and prepregnancy BMI TCD CCD OFD FTD OOD IOD OD

Progress: ultrasound papers (2): ultrasound measures at 4-6 months gestation as predictors of Bayley score U/S Measure 6M MDI (p-value) 12M MDI 6M PDI 12M PDI CCD -- +0.87 (0.02) FTD +0.63 (<0.001) +1.06 (<0.00001) +0.87 (0.001) +0.73 (<0.01) IOD -1.14 (<0.001) -2.00 (<0.0001) -1.58 (<0.001) -1.14 (<0.01)

Other activities Submitted 2 UC Global Health Fellowship applications 1 Ukrainian OB/ultrasonographer for 11 month-fellowship to Describe range of defects diagnosed by prenatal ultrasound in relation to post-natal diagnosis Link to women screened for alcohol and look at prevalence of specific malformations by dose of alcohol reported Describe other malformations in all children in the Eurocat Ukraine register who have an FAS diagnosis 1 U.S. 2nd year medical student from Ukraine for 11 month fellowship to Conduct family interviews with 30 families of an FAS child in Ukraine to describe natural history, recurrence and compare to similar number of families identified through FASD study with high alcohol use but apparently unaffected child Possible collaboration on 2 women in science fellowship applications for K. Sulik