1. Neonatal Screening for Prenatal Alcohol Exposure - Update Joey Gareri HBSc., MSc. Motherisk Laboratory Division of Clinical Pharmacology & Toxicology, Hospital for Sick Children Department of Pharmacology, University of Toronto

2. FASD Diagnosis: Canadian Guidelines (2005) (Chudley et al. 2005) A. Presence of the 3 characteristic facial features (short palpebral fissures, smooth or flattened philtrum, thin vermilion border). B. Evidence of significant prenatal exposure to alcohol at levels known to be associated with physical or developmental effects, or both. C. Presence of 1 or more facial features with growth deficits plus known or probable significant prenatal alcohol exposure. D. Presence of 1 or more facial features with 1 or more central nervous system deficits plus known or probable significant prenatal alcohol exposure. E. Presence of 1 or more facial features with pre- or postnatal growth deficits or both (at the 10th percentile or below [1.5 SD below the mean]) and 1 or more central nervous system deficits plus known or probable significant prenatal alcohol exposure.

3. FASD Diagnosis METHODS: 1)Cranio-facial features 2)Confirmation of in utero alcohol exposure -maternal self-reporting -maternal biomarkers of alcoholism * The use of any single or multiple maternal markers is not very effective in the identification of a drinking mother (Stoler et al., 1998) BIOMARKER SPECIFIC TO PREGNANCY

4. Detecting Alcohol Abuse One standard drink (Canadian definition) One standard drink (Canadian definition) 13.6 grams of ethanol 13.6 grams of ethanol 12 oz. beer (5%) 12 oz. beer (5%) 5 oz. wine (12-15%) 5 oz. wine (12-15%) 1.5 oz. liquor (40%) 1.5 oz. liquor (40%) Alcohol Elimination Rate: ~7 g per hour Alcohol Elimination Rate: ~7 g per hour e.g. 5 drinks in 1 hour (i.e. binge episode) e.g. 5 drinks in 1 hour (i.e. binge episode) 0 BAC within 10 hours 0 BAC within 10 hours 0 UAC within 12 hours 0 UAC within 12 hours

5. Ethanol Metabolism & Elimination

6. FAEE production ETHANOL ADH and Microsomal Oxidation (e.g. CYP 2E1) ACETALDEHYDE FAEE Synthases FAEE Non-Oxidative FATTY ACIDS Oxidative Acyl-coenzyme A:ethanol O-acyltransferase (AEAT) FATTY ACYL CoA POTENTIAL BIOLOGICAL MARKERS

7. The Matrices: FAEE Analysis 1) Neonatal Meconium 2 nd & 3 rd trimester prenatal ethanol exposure 2 nd & 3 rd trimester prenatal ethanol exposure 2) Neonatal Hair 3 rd trimester prenatal ethanol exposure 3 rd trimester prenatal ethanol exposure Chan et al. 2004: FAEE do not cross placenta FAEE do not cross placenta neonatal FAEE = fetal exposure 3) Maternal Hair < 6 month history of general drinking behaviour < 6 month history of general drinking behaviour

8. Meconium FAEE 1) Meconium analysis Begins formation at ~13 weeks of pregnancy Begins formation at ~13 weeks of pregnancy 2 nd & 3 rd trimester exposure 2 nd & 3 rd trimester exposure Available within 72 hours of birth Available within 72 hours of birth Discarded material Discarded material

9. Meconium FAEE: Maternal Alcohol Consumption Bearer et al. 1999: Prospective Study (United States) Bearer et al. 1999: Prospective Study (United States) Ethyl linolate; > 1 drink/week Ethyl linolate; > 1 drink/week N = 248 N = 248 n = 39 confirmed drinkers n = 39 confirmed drinkers Sensitivity 72%; Specificity 51% Sensitivity 72%; Specificity 51% Klein et al. 1999: Case report (Canada) Klein et al. 1999: Case report (Canada) High [FAEE] in meconium w/reported prenatal ethanol consumption High [FAEE] in meconium w/reported prenatal ethanol consumption [FAEE] 34-fold higher than non-drinking control group [FAEE] 34-fold higher than non-drinking control group

10. Meconium FAEE: Maternal Alcohol Consumption Bearer et al. 2003: Prospective study (South Africa) Bearer et al. 2003: Prospective study (South Africa) Ethyl oleate; > 1.5 oz. ethanol/drinking day Ethyl oleate; > 1.5 oz. ethanol/drinking day N = 27 N = 27 n = 21 confirmed drinkers n = 21 confirmed drinkers Sensitivity 84.2%; Specificity 83.3% Sensitivity 84.2%; Specificity 83.3% Chan et al. 2003: Prospective study (Canada, Israel) Chan et al. 2003: Prospective study (Canada, Israel) Meconium [FAEE] baseline = < 2.00 nmol/gram Meconium [FAEE] baseline = < 2.00 nmol/gram n = 206 n = 206 n = 84 non-drinkers; Toronto n = 84 non-drinkers; Toronto n = 99 non-drinkers; Jerusalem n = 99 non-drinkers; Jerusalem n = 17 social drinkers; Toronto n = 17 social drinkers; Toronto n = 6 confirmed drinkers; Toronto n = 6 confirmed drinkers; Toronto Sensitivity 100%; Specificity 98.4% Sensitivity 100%; Specificity 98.4%

11. Meconium FAEE: Population-Based Studies Chan et al. 2003 (Canada) Chan et al. 2003 (Canada) N = 142 meconium samples with suspicion of prenatal exposure N = 142 meconium samples with suspicion of prenatal exposure 71% samples positive for at least one illicit drug 71% samples positive for at least one illicit drug 14% samples positive for FAEE > 2.0 nmol/gram 14% samples positive for FAEE > 2.0 nmol/gram Moore et al. 2003 (United States) Moore et al. 2003 (United States) 2 hospitals: Utah, Hawaii 2 hospitals: Utah, Hawaii Universal anonymous screening Universal anonymous screening N = 725 N = 725 4 th quartile = meconium [FAEE] > 10,000 ng/g 4 th quartile = meconium [FAEE] > 10,000 ng/g

12. Meconium FAEE: Population-Based Studies Gareri et al. in progress (Canada) Gareri et al. in progress (Canada) 5 hospitals: Grey Bruce Region, ON 5 hospitals: Grey Bruce Region, ON Universal anonymous screening Universal anonymous screening N = 683 N = 683 2.5 - 3.5% prevalence of fetal alcohol exposure 2.5 - 3.5% prevalence of fetal alcohol exposure Meconium [FAEE] > 2.0 nmol/g Meconium [FAEE] > 2.0 nmol/g 5-fold > than clinical reporting 5-fold > than clinical reporting Hutson et al. in progress (Uruguay) Hutson et al. in progress (Uruguay) Prospective study; One hospital serving low SES population Prospective study; One hospital serving low SES population N ~900 N ~900 Preliminary results Preliminary results > 30.0% prevalence of fetal alcohol exposure > 30.0% prevalence of fetal alcohol exposure Meconium [FAEE] > 2.0 nmol/g Meconium [FAEE] > 2.0 nmol/g Neonatal outcomes available for comparison Neonatal outcomes available for comparison

13. Meconium FAEE: FASD Outcomes Derauf et al. 2003 (United States) Derauf et al. 2003 (United States) Lower one-minute Apgar scores (p = 0.003) Lower one-minute Apgar scores (p = 0.003) [ethyl oleate] assoc. w/low birth weight (p = 0.006) [ethyl oleate] assoc. w/low birth weight (p = 0.006) N = 422 N = 422 Noland et al. 2003 (United States) Noland et al. 2003 (United States) Decreased score on executive functioning task Decreased score on executive functioning task Tapping inhibition (age 4 years) Tapping inhibition (age 4 years) Lower birth weight, length, head circumference Lower birth weight, length, head circumference N = 316 N = 316 Peterson et al. 2005 (United States) Peterson et al. 2005 (United States) Decreased psychomotor performance (age 2 years; P < 0.04) Decreased psychomotor performance (age 2 years; P < 0.04) N = 202 N = 202

14. Meconium FAEE: FASD Outcomes Jacobson et al. 2006 (South Africa) Jacobson et al. 2006 (South Africa) ↑ [ethyl oleate] in FAS or pFAS diagnosed children ↑ [ethyl oleate] in FAS or pFAS diagnosed children (age 5 years; p < 0.005) (age 5 years; p < 0.005) [ethyl oleate] > maternal self-report correlates to: [ethyl oleate] > maternal self-report correlates to: Recognition memory, Processing speed, Complexity of symbolic play Recognition memory, Processing speed, Complexity of symbolic play N = 55 N = 55 Brien et al. 2006 (Canada) Brien et al. 2006 (Canada) Animal study: guinea pig Animal study: guinea pig ↑ Meconium [FAEE] = ↓ neonatal brain weight ↑ Meconium [FAEE] = ↓ neonatal brain weight N = 51 N = 51 n = 25 ethanol-exposed n = 25 ethanol-exposed n = 23 pair-fed control n = 23 pair-fed control n = 3 water control n = 3 water control

15. Hair FAEE 2) Neonatal Hair Begins formation at ~20 weeks of pregnancy Begins formation at ~20 weeks of pregnancy 3 rd trimester exposure 3 rd trimester exposure Available for up to 3 months after birth Available for up to 3 months after birth Small quantities available Small quantities available 3) Maternal Hair Grows at ~1.0 cm/month Grows at ~1.0 cm/month Contains history of substance use Contains history of substance use

16. Hair FAEE & Maternal Alcohol Consumption Pragst et al. 2001; Wurst et al. 2004 Pragst et al. 2001; Wurst et al. 2004 < 6 cm hair analysis = maximum 6 mos. History < 6 cm hair analysis = maximum 6 mos. History [FAEE] > 1.0 ng/mg [FAEE] > 1.0 ng/mg 75% sensitivity; 100% specificity 75% sensitivity; 100% specificity [FAEE] > 0.5 ng/mg [FAEE] > 0.5 ng/mg 90% sensitivity; 90% specificity 90% sensitivity; 90% specificity Kulaga et al. 2006 Kulaga et al. 2006 Comparison of animal (guinea pig) vs. human data Comparison of animal (guinea pig) vs. human data FAEE incorporation in hair 11-fold higher in humans FAEE incorporation in hair 11-fold higher in humans [ethyl oleate] correlates with total systemic ethanol exposure [ethyl oleate] correlates with total systemic ethanol exposure

17. Hair FAEE: Neonatal Validation Caprara et al. 2005 Caprara et al. 2005 Animal Study (guinea pig) Animal Study (guinea pig) Neonatal [FAEE] 10-fold higher in ethanol-exposed litters Neonatal [FAEE] 10-fold higher in ethanol-exposed litters Caprara et al. 2005 Caprara et al. 2005 Pilot Study; baseline establishment Pilot Study; baseline establishment Community-based pediatric clinic Community-based pediatric clinic N = 56 N = 56 n = 33 non-drinkers n = 33 non-drinkers n = 23 social drinkers (≤ 2 drinks per week) n = 23 social drinkers (≤ 2 drinks per week) Range [FAEE] = 0.00 – 2.95 pmol/mg Range [FAEE] = 0.00 – 2.95 pmol/mg Mean [FAEE] = 0.32 pmol/mg Mean [FAEE] = 0.32 pmol/mg Median [FAEE] = 0.008 pmol/mg Median [FAEE] = 0.008 pmol/mg

18. Future Directions Complete validation of neonatal hair analysis for FAEE Complete validation of neonatal hair analysis for FAEE Baseline establishment in large population Baseline establishment in large population Determine predictive value between [FAEE] and FASD Determine predictive value between [FAEE] and FASD

19. Acknowledgements Canadian Institute for Health Research Dr. Gideon Koren Dr. James Brien Janine Hutson Susan Santiago Dr. Bhushan Kapur THANK YOU THE END

20. Portrait of the Addicted Mother Unemployed (93%) Unemployed (93%) Annual Income < $15,000/yr (CAD) (96%) Annual Income < $15,000/yr (CAD) (96%) Grade 12 education or less (92%) Grade 12 education or less (92%) Single/Divorced/Separated (74%) Single/Divorced/Separated (74%) No permanent residence (23%) No permanent residence (23%) Multiple pregnancies (87%) Multiple pregnancies (87%) Apprehended children (25%) Apprehended children (25%) Children living with other family members (74%) Children living with other family members (74%) Abused by partner (60%) Abused by partner (60%) Depressed (78%) Depressed (78%) Suicidal thinking (25%) Suicidal thinking (25%)

21. OVERVIEW Neonatal Screening for Fetal Alcohol Exposure PROS maximize diagnosis/intervention across socioeconomic lines maximize diagnosis/intervention across socioeconomic lines opportunity to initiate therapy at earliest possible time in development (improved prognosis for outcome) opportunity to initiate therapy at earliest possible time in development (improved prognosis for outcome) avoids marginalization of high-risk women (as opposed to targeted screening) avoids marginalization of high-risk women (as opposed to targeted screening) birth provides a window of opportunity in engaging high-risk women birth provides a window of opportunity in engaging high-risk women optimal intervention timing for behaviour changes in mother optimal intervention timing for behaviour changes in mother can provide adoptive parents with valuable background information can provide adoptive parents with valuable background information enormous research potential in engaging an elusive study population enormous research potential in engaging an elusive study populationCONS potential labeling/stigmatization of mother and child potential labeling/stigmatization of mother and child potential for conflict due to perceived or potential implications of a positive test potential for conflict due to perceived or potential implications of a positive test low disease specificity associated with alcohol exposure (<60% unaffected) low disease specificity associated with alcohol exposure (<60% unaffected) not diagnostic for specific treatment not diagnostic for specific treatment intensive follow-up required, high cost intensive follow-up required, high cost can potentially decrease the likelihood of adoption for exposed infants can potentially decrease the likelihood of adoption for exposed infants

22. Prevention by Intervention NEONATAL INTERVENTION CANNOT PREVENT PRIMARY ALCOHOL-INDUCED DAMAGE Mothers of alcohol-affected children are significantly more likely to produce subsequent alcohol affected children Mothers of alcohol-affected children are significantly more likely to produce subsequent alcohol affected children Substance-addicted women have an 85% incidence of multiple pregnancies (average = 4) and 25% incidence of child apprehension by social services Substance-addicted women have an 85% incidence of multiple pregnancies (average = 4) and 25% incidence of child apprehension by social services EARLY MATERNAL INTERVENTION (e.g. 1 st pregnancy) can potentially prevent future cases of FASD EARLY MATERNAL INTERVENTION (e.g. 1 st pregnancy) can potentially prevent future cases of FASD

23. Prevention by Intervention In FASD In FASD 50-70% incidence of substance addiction 50-70% incidence of substance addiction 50% incidence of inappropriate or promiscuous sexual behaviour 50% incidence of inappropriate or promiscuous sexual behaviour FASD INTERVENTION is capable of alleviating secondary disabilities which perpetuate FASD FASD INTERVENTION is capable of alleviating secondary disabilities which perpetuate FASD